Pharmaceutical compositions comprising thieno[2,3-c]pyridine derivatives and use thereof

ABSTRACT

The present invention provides thieno[2,3-C]pyridine derivatives, pharmaceutical compositions comprising the thieno[2,3-C]pyridine derivatives, and methods of use thereof. The compounds capable of inhibiting glycosaminoglycan (GAG) interactions with effector cell adhesion molecules (ECAM) are useful for treating diseases and disorders mediated by GAG-ECAMs interactions, particularly inflammatory and autoimmune diseases, viral diseases, cancer, and amyloid disorders.

FIELD OF INVENTION

The present invention relates to pharmaceutical compositions comprisingthieno[2,3-c]pyridine compounds capable of inhibiting the interactionsbetween effector cell adhesion molecules (ECAMs), particularlyselectins, and glycosaminoglycans (GAGs), particularly heparan sulfateglycosaminoglycans (HS-GAGs), and to methods for the treatment orprevention of diseases or disorders related to cell adhesion and cellmigration, particularly for the treatment or prevention of inflammatoryand autoimmune diseases and disorders, as well as of viral diseases,cancer, and amyloid disorders.

BACKGROUND OF THE INVENTION

The inflammatory response is mediated primarily by leukocytes,neutrophils and lymphocytes, which circulate in the blood and reversiblyinteract with the vascular endothelium. In response to inflammatorystimuli, the leukocytes adhere tightly to the vascular endothelium,migrate (extravasate) through the vessel wall, and subsequently movealong a chemotactic gradient toward the inflammatory stimulus. Theinteraction of leukocytes with vascular endothelial cells is thus anessential initial step in the inflammatory response. Selectins play akey role in inflammation, as they are responsible for the initialattachment of blood borne leukocytes to the vasculature. Preventingselectin-mediated cell adhesion can ameliorate or circumvent thedeleterious consequences of inflammation. Therefore, selectins are theprime target for the therapy of cell-adhesion disorders, specificallyfor treatment of inflammation.

Selectins regulate neutrophil and lymphocyte adhesion to and entry intolymphoid tissues and sites of inflammation (Rosen, Am. J. Respir. Cell.Mol. Biol., 3:397-402, 1990). The three known selectins are E-selectin(formerly known as ELAM.1), P-selectin (formerly known as PADGEM,GMP-140, or CD61) and L-selectin (formerly known as mLHR, Leu8, TQ-1,gp90, MEL, Lam-1, or Lecam-1) (Lasky, Annu. Rev. Biochem. 64:113, 1995;Kansas, Blood 88:3259, 1996). Each selectin is regulated differently,and participates in a different manner in the process of inflammation orimmunity. The lectin domains of each selectin are critical to theadhesive functions of the proteins. The selectins are responsible forleukocyte capture from the blood stream and mediate their intermittentattachment with consequent leukocyte “rolling” along the endothelialcell surface. This capture allows the cascade of secondary, tightercell-adhesive events to take place. L-selectin is constitutivelyexpressed by leukocytes and mediates lymphocyte adhesion to peripherallymph node high endothelial venules, and neutrophil adhesion tocytokine-activated endothelial cells (Spertini et al. J. Immunol.147:2565-2573, 1991). In inflammatory disorders it may be L-selectinthat plays the most significant role (Shimizu et al., Immunol. Today13:106, 1992; Picker et al., Annu. Rev. Immunol. 10:561, 1992).

Buerke et al. demonstrated the important role of selectins ininflammatory states such as ischemia-reperfusion injury in cats (Buerke,M. et al., J. Clin. Invest. 93:1140,1994). The presence of L-selectinand E- or P-selectin ligands on mononuclear cells has implicated thesereceptor-ligand interactions in chronic inflammation. (L. Lasky Annu.Rev. Biochem. 64:113-39, 1995). Monoclonal antibodies to L-selectinprevent neutrophil emigration into inflamed skin (Lewinsohn et al., J.Immunol. 138:4313, 1987), neutrophil and monocyte emigration intoinflamed ascites (Jutila et al., J. Immunol. 143:3318, 1989), andneutrophil emigration into inflamed peritoneum. Jasin et al. providesupport for the use of antibodies in inhibiting neutrophil accumulationin inflamed synovium (Jasin et al., Arthritis Rheum. 33:S34, 1990).Monoclonal antibody EL-246, directed against both L-selectin andE-selectin, attenuated sepsis-induced lung injury (Ridings, P C et al.,Arch Surg. 1199-1208, 1995). Monoclonal antibody SMART is an L-selectinblocking antibody that is being used in clinical trials for traumaassociated with multiple organ failure (this condition is believed to bedue in part to infiltration of inflammatory cells). The anti-L-selectinantibody is presumed to provide its therapeutic effect by preventingneutrophil adhesion to endothelium and it is active in vivo in a primatemodel of severe trauma (Schlag G et al, Critical Care Medicine 1999, 27,1900-1907). It is believed that this monoclonal antibody will be alsouseful in the treatment of adult respiratory distress syndrome andmyocardial infarction.

Glycosaminoglycans (also referred to herein as “GAG” or “GAGs”) arenaturally-occurring carbohydrate-based molecules implicated in theregulation of a number of cellular processes, including bloodcoagulation, angiogenesis, tumor growth, and smooth muscle cellproliferation, most likely by interaction with effector molecules. GAGsare often, but not always, found covalently bound to protein cores instructures called proteoglycans. Proteoglycan structures are abundant oncell surfaces and are associated with the extracellular matrix aroundcells. GAGs consist of repeating disaccharide units. For example,heparan sulfate glycosaminoglycans (also referred to herein as“HS-GAGs”) consist of repeating disaccharide units of D-glucuronic acidand N-acetyl- or N-sulfo-D-glucosamine. The high molecular diversity ofHS-GAGs is due to their unique sulfation pattern (Sasisekharan, R. andVenkataraman, G., Current Opinion in Chem. Biol., 4, 626-631, 2000;Lindahl, U. et al., J. Biol. Chem., 273, 24979-24982, 1998; Esko, J. andSelleck, S. B., Annu. Rev. Biochem., 71, 435-471, 2002). One of the mostthoroughly studied HS-GAGs is the widely used anticoagulant heparin.Heparin is a highly sulfated form of heparan sulfate found in mastcells. Many important regulatory proteins including cytokines, growthfactors, enzymes, and cell adhesion molecules bind tightly to heparin.Although interactions of proteins with GAGs such as heparin and heparansulfate are of great biological importance, the structural requirementsfor protein-GAG binding have not been well characterized. Ionicinteractions are important in promoting protein-GAG binding and thespacing of the charged residues may determine protein-GAG affinity andspecificity.

The HS-GAG paradigm provides new approaches and strategies fortherapeutic intervention at the cell-tissue-organ interface. Forexample, identification of specific HS-GAG sequences that affectparticular biological processes will enable the development of novelmolecular therapeutics based on polysaccharide sequence. SyntheticHS-GAGs, or molecular mimics of HS-GAG sequences, may provide newapproaches for combating health problems such as bacterial and viralinfections, atherosclerosis, cancer, and Alzheimer's disease.

Selectins mediate their adhesive functions via lectin domains that bindto carbohydrate ligands. Emerging evidence indicates that GAGs, and inparticular HS-GAGs, are carbohydrate receptors with which the selectinsinteract (Nelson R M, et al., Blood 82, 3253-3258, 1993; Ma, Y Q andGeng, J G, J. Immunol. 165, 558-565, 2000; Giuffre, L. et al., J. Cell.Biol. 136, 945-956, 1997; Watanabe N., et al., J. Biochem. 125, 826-831,1999; Li Y F et al., FEBS Lett 444, 201-205, 1999). Consistent with thisobservation, heparin, HS-GAG and heparin-derived oligosaccharides blockL-selectin-dependent adhesion directly (U.S. Pat. No. 5,527,785 toBevilacqua et al.). Furthermore, short sulfated heparin-derivedtetrasaccharides reduced binding of neutrophils to COS cells expressingP-selectin (Nelson R M, et al., Blood 82, 3253-3258, 1993). Themultivalent nature of HS may be an important factor in bindingL-selectin under flow conditions (Sanders et al, ibid).

As the interactions between GAGs and selectins play an important role incell-matrix and cell-cell adhesion, the latter are processes involved incertain diseases and inflammatory disorders, modulating theseinteractions have therapeutic implications.

Bevilacqua et al (U.S. Pat. No. 5,527,785) provide a method ofmodulating selectin binding in a subject by administering heparin-likeoligosaccharides. The oligosaccharides act by binding to L- orP-selectin.

Xie X et al (JBC 275, 34818-25, 2000) described inhibition of L- andP-selectin mediated cell adhesion by sulfated saccharides, includingcarboxyl-reduced and sulfated heparin. While these molecules have beenuseful to show the utility of selectin blockers for treatinginflammation, each has significant drawbacks as a therapeutic, includingshort in vivo half-life, high cost, potential immunogenicity, and otherpossible side effects. A further limitation of these approaches is lackof efficient means to improve the pharmacological properties of thesemolecules.

In addition, several groups developed small peptides with highaffinities for heparin or for heparin-like molecules (i.e., PGs, orother GAGs) (see, for example, U.S. Pat. No. 5,919,761 to Wakefield etal.) to use in a variety of applications for modulating the activitiesof native GAGs and PGs.

There is still an unmet need to have a non-peptide, small syntheticcompounds, which are capable of modulating the functions of GAGs and theinteractions between GAGs and GAG effector protein molecules.

U.S. Pat. 6,232,320 discloses the use of thieno[2,3-c]pyridines asinhibitors of cell adhesion useful as inhibitors of inflammation. Thedisclosed compounds are different from the compounds of the presentinvention as they posses a different heterocyclic system and do notposses sulfonylbenzoylamino group.

Japanese Patent Application JP 2001151779 discloses4,5,6,7-tetrahydrothieno[2,3-c]pyridines, pharmaceutical compositions,and TNF-α formation inhibitors containing them, also disclosed in FujitaM, et al. (Bioorg. Med. Chem. Lett., 12: 1607-1611, 2002). A relatedJapanese Patent Application JP 2001151780 discloses novel4,5,6,7-tetrahydrothieno[2,3-c]pyridines as inhibitors of TNF-alphasynthesis, also disclosed in Fujita et al. (Bioorg. Med. Chem. Lett. 12:1897-1900, 2002). The two Japanese Patent Application provide differentsubstituents in position 3 of the 4,5,6,7-tetrahdrothieno [2,3-c]pyridine ring (arylcarbonyl in the 2001151779 Application and carboxy oralkoxycarbonyl in the 2001151780 Application). Al of these compounds,however, are different from those of the present invention as they donot contain a sulfonylbenzoylamino group as part of the scaffold, anessential feature of the compounds of the present invention.

Balakin et al. (J. Chem. Inf. Comput. Sci. 42: 1332-1342, 2002) describein silico property-based design of a G-protein coupled receptor(GPCR)-targeted library of compounds. Among the tens of thousands ofstructures screened in silico, certain compounds from the GPCR-targetedlibrary including a single thieno [2,3-c]pyridine compound weredesignated the highest scoring structures by the selection criteriaapplied in that analysis.

SciFinder Scholar database, release 2003, lists 2846 derivatives (as ofDec. 30, 2003) of thieno[2,3-c]pyridine, but no utility is attributed toany of these compounds and no chemical synthesis data are described.

Chemical Diversity Labs Inc. (San Diego, Calif.), a supplier of chemicalcompounds, released a database named CombiLab Probe Libraries (June 2002revision; 220,674 compound structures), which lists 438 derivatives ofthieno[2,3-c] pyridine, but no utility or chemical synthesis data isdescribed.

I.F. Lab (Kiev, Ukraine), a supplier of chemical compounds, released adatabase named IF LAB Libraries (July 2003; 77,098 compound structures),which lists 3145 derivatives of thieno[2,3-c] pyridine, but no utilityor chemical synthesis data is described. Some of the compounds inChemical Diversity Labs Inc. database are the same as in I.F. LABdatabase.

Nowhere in the background art is it taught or suggested thatsulfonylbenzoylamino derivatives of thieno [2,3-c] pyridines havebeneficial pharmaceutical activities.

SUMMARY OF THE INVENTION

It is an object of some aspects of the present invention to providepharmaceutical compositions comprising small organic compounds formedical and diagnostic use, wherein the small organic compounds areinhibitors of the interactions between effector cell adhesion molecules(ECAMs), specifically L-selectin and P-selectin, with glycosaminoglycans(GAGs), specifically heparan sulfate glycosaminoglycans (HS-GAGs).Accordingly, these compositions are useful as inhibitors of cell-cellinteractions mediated by L-selectin and P-selectin, particularlyleukocyte adhesion, migration and infiltration. In addition, thecompositions interact directly with HS-GAGs and are therefore useful asinhibitors of any HS-GAG mediated processes and conditions.

According to one aspect, the present invention provides a pharmaceuticalcomposition comprising as an active ingredient a compound of the generalformula I:

wherein:

R¹ is selected from the group consisting of H; straight or branchedalkyl of 1-6 carbon atoms; arylalkyl; substituted arylalkyl; cycloalkyl,optionally substituted with alkyl groups; alkanoyl; arylcarbonyloptionally substituted at the aryl group; cycloalkylcarbonyl;alkoxycarbonyl;

R₂ is selected from the group consisting of carboxy; cyano;aminocarbonyl; alkylaminocarbonyl; arylaminocarbonyl optionallysubstituted at the aryl group; dialkylaminocarbonyl wherein each alkylis straight or branched chain C₁-C₆ alkyl or both alkyl groups togethermay form a 3-7 membered saturated, unsaturated or aromatic monocyclic orbicyclic nitrogen containing heterocyclyl, optionally containing one ortwo additional heteroatoms; allcoxycarbonyl; alkanoyl;cycloalkylcarbonyl; arylcarbonyl optionally substituted on the arylgroup, benzothiazol-2-yl;

R₃ and R₄ are selected from the group consisting of C₁-C₆ alkyl,optionally substituted by hydroxy, alkoxy, amino or alkylamino, C₂-C₄monounsaturated alkenyl, cycloalkyl, aryl, arylmethyl, or R₃ and R4together may form an optionally substituted 5-7 membered saturated,unsaturated or aromatic monocyclic or bicyclic nitrogen containingheterocyclyl, optionally containing one or two additional heteroatoms;

R₅, R₆, R₇ and R₈ are selected from the group consisting of H or C₁-C₆alkyl, with the proviso that when R₅, R₆, R₇ and R8 are C₁-C₆ alkyl, R₁is hydrogen;

and pharmaceutically acceptable salts thereof; further comprising apharmaceutically acceptable diluent or carrier.

According to one embodiment, R₁ is selected from the group consisting ofmethyl, ethyl, 1-methylethyl, phenylmethyl, acetyl, ethoxycarbonyl andR₅═R₆═R₇═R₈ are hydrogen.

According to another embodiment, R₁ is hydrogen and R₅═R₆═R₇═R₈ arehydrogens or methyl groups.

According to yet another embodiment, R₁═R₅═R₆ are methyl and R₇═R₈ arehydrogens.

According to another embodiment, R₂ is selected from the groupconsisting of cyano, methoxycarbonyl, ethoxycarbonyl, aminocarbonyl,methylaminocarbonyl, dimethylaminocarbonyl, pyrrolidinylcarbonyl,piperidinylcarbonyl, morpholinylcarbonyl, benzothiazol-2-yl.

According to further embodiment, R₃ and R4 are selected from the groupconsisting of methyl, ethyl, propyl, butyl, methoxyethyl, chlorobutyl,cyanoethyl, phenyl, cyclopentyl, cyclohexyl, phenylmethyl, allyl orcrotyl, R₃ and R₄ may be equal or different.

According to another embodiment, R₃ and R₄ form pyrrolidine, piperidine,2-methyl, 3-methyl, 4-methyl or 3,5-dimethyl piperidine,perhydroazepine, morpholine, piperazine, 4-methylpiperazine,3,4-dihydro-2(1H)-isoquinolinyl, 3,4-dihydro-1(2H)quinoline,1,3,3-trimethyl-6-azabicyclo[3.2.1]oct-6-ane and substituted derivativesthereof. The substituted derivatives include, but are not limited to,piperazinyl-4-carboxylic acid ester, piperidinyl-4-carboxylic acidester, piperidinyl-3-carboxylic acid ester.

According to certain preferred embodiments, the present inventionprovides compositions comprising compounds of formula I selected from:

2-[[4-[(ethylbutylamino)sulfonyl]benzoyl]amino]-3-(benzothiazol-2-yl)-6-ethyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine(Compound no. 5);

2-[[(4-(3,4-dihydro-2(1H)-isoquinolinyl)sulfonyl]benzoyl]amino]-6-(1-methylethyl)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxamide(Compound no.1);

2-[[4-(methylphenylamino)sulfonyl]benzoyl]amino]-6-(1-methylethyl)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxamide;

2-[[4-(3,4-dihydro-1(2H)-quinolinyl)sulfonyl]benzoyl]amino]-4,5,6,7-tetrahydro-5,5,7,7-tetramethylthieno[2,3-c]pyridine-3-carboxamide;

2-[[4-[(diethylamino)sulfonyl]benzoyl]amino]-3-(benzothiazol-2-yl)-6-ethyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine(Compound no. 11);

2-[[4-(morpholinylsulfonyl)benzoyl]amino]-3-(benzothiazol-2-yl)-6-(1-methylethyl)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine;

2-[[4-(diethylamio)sulfonyl]benzoyl]amino]-4,5,6,7-tetrahydro-5,5,7,7-tetramethylthieno[2,3-c]pyridine-3-carboxylic acid ethyl ester;

2-[[4-(3,4-dihydro-1(2H)-quinolinyl)sulfonyl]benzoyl]amino]-3-(benzothiazol-2-yl)-4,5,6,7-tetrahydro-5,5,7,7-tetramethylthieno[2,3-c]pyridine;

2-[[4-(hexahydro-1H-azepin-1-yl)sulfonyl]benzoyl]amino]-4,5,6,7-tetrahydro-5,5,7,7-tetramethylthieno[2,3-c]pyridine-3-carboxylic acid ethyl ester;

2-[[4-[[4-(methyl)-1-piperazinyl]sulfonyl]benzoyl]amino]-3-(benzothiazol-2-yl)-6-methyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine;

2-[[4-[(1,3,3-trimethyl-6-azabicyclo[3.2.1]oct-6-yl)sulfonyl]benzoyl]amino]-3-(benzothiazol-2-yl)-6-methyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine;

2-[[4-[(methylphenylamino)sulfonyl]benzoyl]amino]-3-(benzothiazol-2-yl)-6-methyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine;

2-[[4-(morpholinylsulfonyl)benzoyl]amino]-3-(benzothiazol-2-yl)-6-methyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine;

2-[[4-[(diethylamino)sulfonyl]benzoyl]amino]-3-(benzothiazol-2-yl)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-6-carboxylicacid ethyl ester;

2-[[4-[[4-(3-methyl-1-piperidinyl)]sulfonyl]benzoyl]amino]-3-(benzothiazol-2-yl)-6-methyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine;

2-[[4-[(diethylamino)sulfonyl]benzoyl]amino]-3-(benzothiazol-2-yl)-6-(phenylmethyl)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine;

2-[[4-[(diethylamino)sulfonyl]benzoyl]amino]-3-(benzothiazol-2-yl)-6-methyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine;

2-[[4-[[4-(ethoxycarbonyl)-1-piperazinyl]sulfonyl]benzoyl]amino]-4,5,6,7-tetrahydro-5,5,7,7-tetramethylthieno[2,3-c]pyridine-3-carboxamide;

2-[[4-[(cyclohexylmethylamino)sulfonyl]benzoyl]amino]-4,5,6,7-tetrahydro-5,5,7,7-tetramethylthieno[2,3-c]pyridine-3-carboxamide;

2-[[4-[(di-2-propenylamino)sulfonyl]benzoyl]-4,5,6,7-tetrahydro-5,5,7,7-tetramethylthieno[2,3-c]pyridine-3-carboxylicacid methyl ester;

2-[[4-[(di-2-methoxyethylamino)]sulfonyl]benzoyl]-4,5,6,7-tetrahydro-5,5,7,7-tetramethylthieno[2,3-c]pyridine-3-carboxamide;

2-[[4-[(1,3,3-trimethyl-6-azabicyclo[3.2.1.]oct-6-yl)sulfonyl]benzoyl]amino]-6-methyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxamide;

2-[[4-[(diethylamino)sulfonyl]benzoyl]amino]-3-(benzothiazol-2-yl)-6-methyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine;

2-[[4-[(diethylamino)sulfonyl]benzoyl]amino]-3-(benzothiazol-2-yl)-6-(1-methylethyl)4,5,6,7-tetrahydrothieno[2,3-c]pyridine;

2-[[4-[(di-2-methoxyethylamino)sulfonyl]benzoyl]-amino]-3-(benzothiazol-2-yl)-6-methyl4,5,6,7-tetrahydrothieno[2,3-c]pyridine;

2-[[4-[(methylphenylamino)sulfonyl]benzoyl]amino]-3-(benzothiazol-2-yl)-6-methyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine;

2-[[4-[[4-(ethoxycarbonyl)-1-piperazinyl]sulfonyl]benzoyl]amino]-3-(benzothiazol-2-yl)-6-methyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine;

2-[[4-[(methylbutylamino)sulfonyl]benzoyl]amino]-6-(1-methylethyl)4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxylicacid ethyl ester;

2-[[4-[[4-(ethoxycarbonyl)-1-piperazinyl]sulfonyl]benzoyl]amino]-6-(1-methylethyl)4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxylicacid ethyl ester;

2-[[4-(diethylamino)sulfonyl]benzoyl]amino]-4,5,6,7-tetrahydro-5,5,7,7-tetramethylthieno[2,3-c]pyridine-3-carboxamide;

2-[[4-[(methylphenylamino)sulfonyl]benzoyl]amino]-6-ethyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxylicacid methylamide;

2-[[4-[[ethyl(phenylmethyl)amino]sulfonyl]benzoyl]amino]-6-ethyl4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxamide;

2-[[4-[(4-methyl-1-piperazinyl)sulfonyl]benzoyl]amino]-6-ethyl4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxamide;

2-[[(4-(3,4-dihydro-1(2H)-quinolinyl)sulfonyl]benzoyl]amino]-6-ethyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxylicacid methylamide;

2-[[4-[(4-methyl-1-piperazinyl)sulfonyl]benzoyl]amino]-6-ethyl-4,5,6,7-tetrahydrothieno[2,3-c)pyridine-3-carboxylicacid methylamide;

2-[[4-[(4-methyl-1-piperazinyl)sulfonyl]benzoyl]amino]-3-(benzothiazol-2-yl)-6-ethyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine;

2-[[4-(diethylamino)sulfonyl]benzoyl]amino]-6-ethyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxylicacid N-methylamide;

2-[[4-(diethylamino)sulfonyl]benzoyl]amino]-6-ethyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxylicacid morpholinylamide.

According to currently more preferred embodiments, the pharmaceuticalcomposition of the invention comprise a compound selected from:

2-[[4-[(ethylbutylamino)sulfonyl]benzoyl]amino]-3-(benzothiazol-2-yl)-6-ethyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine(Compound no. 5);

2-[[4-[(diethylamino)sulfonyl]benzoyl]amino]-3-(benzothiazol-2-yl)-6-ethyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine(Compound no. 11);

2-[[4-[[ethyl(phenylmethyl)amino]sulfonyl]benzoyl]amino]-6-ethyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxamide(Compound no. 28);

2-[[4-[(4-methyl-1-piperazinyl)sulfonyl]benzoyl]amino]-6-ethyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxamide(Compound 29).

According to another embodiment the pharmaceutical compositions of thepresent invention inhibit the binding of GAGs to GAG-specific-ECAMs.

According to a further embodiment the pharmaceutical compositions of thepresent invention inhibit the interactions of HS-GAGs with selectins,specifically L-selectin and P-selectin.

According to yet another embodiment, the pharmaceutical compositions ofthe present invention bind directly to GAGs, specifically HS-GAG.

According to a further embodiment, the pharmaceutical compositions ofthe present invention inhibit leukocyte and neutrophil infiltration invivo.

It is to be understood that the present invention does not encompass anycompounds or pharmaceutical compositions thereof for which such apharmaceutical activity has been disclosed. Explicitly excluded is thecompound2-[[4-[(1,3,3-trimethyl-6-azabicyclo[3.2.1.]oct-6-yl)sulfonyl]benzoyl]amino]-6-ethyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxylicacid ethyl ester.

According to another aspect the present invention discloses certainnovel compounds and these are claimed as such. According to oneembodiment, the present invention provides a small organic compoundhaving the formula2-[[4-[[ethyl(phenylmethyl)amino]sulfonyl]benzoyl]amino]-6-ethyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxamide(Compound No. 28).

According to another embodiment, the present invention provides a smallorganic compound having the formula2-[[4-[(4-methyl-1-piperazinyl)sulfonyl]benzoyl]amino]-6-ethyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxamide(Compound No. 29).

According to yet another aspect, the present invention provides a methodfor inhibiting cell adhesion and cell migration in vitro comprising thestep of exposing the cells to a pharmaceutical composition according tothe invention in an amount sufficient to inhibit binding of GAGs to GAGspecific ECAMs.

According to yet other aspect, the present invention provides a methodfor the treatment or prevention of diseases and disorders related tocell adhesion and cell migration mediated by GAG-ECAM interactions,comprising the step of administering to a subject in need thereof atherapeutically effective amount of a pharmaceutical compositioncomprising a compound of the general formula I:

wherein:

R₁ is selected from the group consisting of H; straight or branchedalkyl of 1-6 carbon atoms; arylalkyl; substituted arylalkyl; cycloalkyl,optionally substituted with alkyl groups; alkanoyl; arylcarbonyloptionally substituted at the aryl group; cycloalkylcarbonyl;alkoxycarbonyl;

R₂ is selected from the group consisting of carboxy; cyano;aminocarbonyl; alkylaminocarbonyl; arylaminocarbonyl optionallysubstituted at the aryl group; dialkylaminocarbonyl wherein each alkylis straight or branched chain C₁-C₆ alkyl or both alkyl groups togethermay form a 3-7 membered saturated, unsaturated or aromatic monocyclic orbicyclic nitrogen containing heterocyclyl, optionally containing one ortwo additional heteroatoms; alkoxycarbonyl; alkanoyl;cycloalkylcarbonyl; arylcarbonyl optionally substituted on the arylgroup, benzothiazol-2-yl;

R₃ and R₄ are selected from the group consisting of C₁-C₆ alkyl,optionally substituted by hydroxy, alkoxy, amino or alkylamino, C₂-C₄monounsaturated alkenyl, cycloalkyl, aryl, arylmethyl, or R₃ and R₄together may form an optionally substituted 5-7 membered saturated,unsaturated or aromatic monocyclic or bicyclic nitrogen containingheterocyclyl, optionally containing one or two additional heteroatoms;

R₅, R₆, R₇ and R₈ are selected from the group consisting of H or C₁-C₆alkyl, with the proviso that when R₅, R₆, R₇ and R₈ are C₁-C₆ alkyl, R₁is hydrogen;

and pharmaceutically acceptable salts thereof; further comprising apharmaceutically acceptable diluent or carrier.

According to certain preferred embodiments, the method for the treatmentor prevention of diseases and disorders related to cell adhesion andcell migration mediated by GAG-ECAM interactions comprise the step ofadministering to a subject in need thereof a therapeutically effectiveamount of a pharmaceutical composition according to the inventioncomprising as an active ingredient a compound of formula I selectedfrom:

2-[[4-[(ethylbutylamino)sulfonyl]benzoyl]amino]-3-(benzothiazol-2-yl)-6-ethyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine(Compound no. 5);

2-[[(4-(3,4-dihydro-2(1H)-isoquinolinyl)sulfonyl]benzoyl]amino]-6-(1-methylethyl)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxamide(Compound no.1);

2-[[4-(methylphenylamino)sulfonyl]benzoyl]amino]-6-(1-methylethyl)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxamide;

2-[[4-(3,4-dihydro-1(2H)-quinolinyl)sulfonyl]benzoyl]amino]-4,5,6,7-tetrahydro-5,5,7,7-tetramethylthieno[2,3-c]pyridine-3-carboxamide;

2-[[4-[(diethylamino)sulfonyl]benzoyl]amino]-3-(benzothiazol-2-yl)-6-ethyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine(Compound no. 11);

2-[[4-(morpholinylsulfonyl)benzoyl]amino]-3-(benzothiazol-2-yl)-6-(1-methylethyl)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine;

2-[[4-(diethylamino)sulfonyl]benzoyl]amino]-4,5,6,7-tetrahydro-5,5,7,7-tetramethylthieno[2,3-c]pyridine-3-carboxylic acid ethyl ester;

2-[[4-(3,4-dihydro-1(2H)-quinolinyl)sulfonyl]benzoyl]amino]-3-(benzothiazol-2-yl)-4,5,6,7-tetrahydro-5,5,7,7-tetramethylthieno[2,3-c]pyridine;

2-[[4-(hexahydro-1H-azepin-1-yl)sulfonyl]benzoyl]amino]-4,5,6,7-tetrahydro-5,5,7,7-tetramethylthieno[2,3-c]pyridine-3-carboxylic acid ethyl ester;

2-[[4-[[4-(methyl)-1-piperazinyl]sulfonyl]benzoyl]amino]-3-(benzothiazol-2-yl)-6-methyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine;

2-[[4-[(1,3,3-trimethyl-6-azabicyclo[3.2.1]oct-6-yl)sulfonyl]benzoyl]amino]-3-(benzothiazol-2-yl)-6-methyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine;

2-[[4-[(methylphenylamino)sulfonyl]benzoyl]amino]-3-(benzothiazol-2-yl)-6-methyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine;

2-[[4-(morpholinylsulfonyl)benzoyl]amino]-3-(benzothiazol-2-yl)-6-methyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine;

2-[[4-[(diethylamino)sulfonyl]benzoyl]amino]-3-(benzothiazol-2-yl)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-6-carboxylicacid ethyl ester;

2-[[4-[[4-(3-methyl-1-piperidinyl)]sulfonyl]benzoyl]amino]-3-(benzothiazol-2-yl)-6-methyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine;

2-[[4-[(diethylamino)sulfonyl]benzoyl]amino]-3-(benzothiazol-2-yl)-6-(phenylmethyl)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine;

2-[[4-[(diethylamino)sulfonyl]benzoyl]amino]-3-(benzothiazol-2-yl)-6-methyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine;

2-[[4-[[4-(ethoxycarbonyl)-1-piperazinyl]sulfonyl]benzoyl]amino]-4,5,6,7-tetrahydro-5,5,7,7-tetramethylthieno[2,3-c]pyridine-3-carboxamide;

2-[[4-[(cyclohexylmethylamino)sulfonyl]benzoyl]amino]-4,5,6,7-tetrahydro-5,5,7,7-tetramethylthieno[2,3-c]pyridine-3-carboxamide;

2-[[4-[(di-2-propenylamino)sulfonyl]benzoyl]-4,5,6,7-tetrahydro-5,5,7,7-tetramethylthieno[2,3-c]pyridine-3-carboxylicacid methyl ester;

2-[[4-[(di-2-methoxyethylamino)]sulfonyl]benzoyl]-4,5,6,7-tetrahydro-5,5,7,7-tetramethylthieno[2,3-c]pyridine-3-carboxamide;

2-[[4-[(1,3,3-trimethyl-6-azabicyclo[3.2.1.]oct-6-yl)sulfonyl]benzoyl]amino]-6-methyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxamide;

2-[[4-[(diethylamino)sulfonyl]benzoyl]amino]-3-(benzothiazol-2-yl)-6-methyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine;

2-[[4-[(diethylamino)sulfonyl]benzoyl]amino]-3-(benzothiazol-2-yl)-6-(1-methylethyl)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine;

2-[[4-[(di-2-methoxyethylamino)sulfonyl]benzoyl]-amino]-3-(benzothiazol-2-yl)-6-methyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine;

2-[[4-[(methylphenylamino)sulfonyl]benzoyl]amino]-3-(benzothiazol-2-yl)-6-methyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine;

2-[[4-[[4-(ethoxycarbonyl)-1-piperazinyl]sulfonyl]benzoyl]amino]-3-(benzothiazol-2-yl)-6-methyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine;

2-[[4-[(methylbutylamino)sulfonyl]benzoyl]amino]-6-(1-methylethyl)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxylicacid ethyl ester;

2-[[4-[[4-(ethoxycarbonyl)-1-piperazinyl]sulfonyl]benzoyl]amino]-6-(1-methylethyl)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxylicacid ethyl ester;

2-[[4-(diethylamino)sulfonyl]benzoyl]amino]-4,5,6,7-tetrahydro-5,5,7,7-tetramethylthieno[2,3-c]pyridine-3-carboxamide;

2-[[4-[(methylphenylamino)sulfonyl]benzoyl]amino]-6-ethyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxylicacid methylamide;

2-[[4-[[ethyl(phenylmethyl)amino]sulfonyl]benzoyl]amino]-6-ethyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxamide;

2-[[4-[(4-methyl-1-piperazinyl)sulfonyl]benzoyl]amino]-6-ethyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxamide;

2-[[(4-(3,4-dihydro-1(2H)-quinolinyl)sulfonyl]benzoyl]amino]-6-ethyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxylicacid methylamide;

2-[[4-[(4-methyl-1-piperazinyl)sulfonyl]benzoyl]amino]-6-ethyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxylicacid methylamide;

2-[[4-[(4-methyl-1-piperazinyl)sulfonyl]benzoyl]amino]-3-(benzothiazol-2-yl)-6-ethyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine;

2-[[4-(diethylamino)sulfonyl]benzoyl]amino]-6-ethyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxylicacid N-methylamide;

2-[[4-(diethylamino)sulfonyl]benzoyl]amino]-6-ethyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxylicacid morpholinylamide.

According to currently more preferred embodiments, the method for thetreatment or prevention of diseases and disorders related to celladhesion and cell migration mediated by GAG-ECAM interactions compriseadministering to a subject in need thereof a therapeutically effectiveamount of a pharmaceutical composition comprising as an activeingredient a compound of formula I selected from:

2-[[4-[(ethylbutylamino)sulfonyl]benzoyl]amino]-3-(benzothiazol-2-yl)-6-ethyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine(Compound no. 5);

2-[[4-[(diethylamino)sulfonyl]benzoyl]amino]-3-(benzothiazol-2-yl)-6-ethyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine(Compound no. 11);

2-[[4-[[ethyl(phenylmethyl)amino]sulfonyl]benzoyl]amino]-6-ethyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxamide(Compound no. 28);

2-[[4-[(4-methyl-1-piperazinyl)sulfonyl]benzoyl]amino]-6-ethyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxamide(Compound no. 29).

According to one embodiment, the invention provides a method fortreatment or prevention of diseases or disorders mediated by GAG-ECAMinteractions, wherein the GAGs are selected from the group consisting ofheparan sulfate (HS-GAG), heparin, chondroitin sulfate, dermatansulfate, keratan sulfate, and derivatives and fragments thereof.According to a currently preferred embodiment, the GAG is HS-GAG.

According to yet another embodiment, the invention provides a method fortreatment or prevention of diseases or disorders mediated by GAG-ECAMinteractions, wherein the GAG specific ECAMs are selected from the groupconsisting of selecting, integrins, fibronectin, and cytokines.According to a currently preferred embodiment, the GAG specific ECAMsare selected from the group consisting of L-selectin and P-selectin.

According to another embodiment, the disease or disorder mediated byGAG-ECAM interactions is selected from inflammatory processes ordisorders, autoimmune processes or diseases, platelet-mediatedpathologies, tumor metastasis, viral diseases, coagulation disorders,atherosclerosis, amyloid disorders, and kidney diseases.

According to another embodiment, the inflammatory processes or disordersmediated by GAG-ECAM interactions are exemplified by, but not restrictedto, septic shock, post-ischemic leukocyte-mediated tissue damage,frost-bite injury or shock, acute leukocyte-mediated lung injury, acutepancreatitis, nephritis, asthma, traumatic shock, stroke, traumaticbrain injury, nephritis, acute and chronic inflammation, includingatopic dermatitis, psoriasis, uveitis, retinitis, and inflammatory boweldisease.

According to a currently preferred embodiment, the invention provides amethod for the prevention or treatment of inflammatory bowel diseasecomprising the step of administering to a subject in need thereof atherapeutically effective amount of a pharmaceutical compositioncomprising a thieno[2,3-c]pyridine compound of the formula2-[[4-[(diethylamino)sulfonyl]benzoyl]amino]-3-(benzothiazol-2-yl)-6-ethyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine(Compound no. 11) and a pharmaceutically acceptable salt thereof,further comprising a pharmaceutically acceptable diluent or carrier.

According to yet another embodiment, the autoimmune diseases mediated byGAG-ECAM interactions are exemplified by, but not restricted to,rheumatoid arthritis and multiple sclerosis.

According to a currently preferred embodiment, the invention provides amethod for the prevention or treatment of multiple sclerosis comprisingthe step of administering to a subject in need thereof a therapeuticallyeffective amount of a pharmaceutical composition comprising as an activeingredient thieno[2,3-c]pyridine compound2-[[4-[(diethylamino)sulfonyl]benzoyl]amino]-3-(benzothiazol-2-yl)-6-ethyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine(Compound no. 11) and a pharmaceutically acceptable salt thereof;further comprising a pharmaceutically acceptable carrier or diluent.

According to yet further embodiment, the diseases or disorders mediatedby GAG-ECAM interactions include those mediated by cell-cell,cell-virus, cell-matrix, and cell-protein interactions, exemplified by,but not restricted to, virus attachment to a cell, cell adhesion,platelet aggregation, lymphocyte adhesion and migration, and amyloidfibril formation.

According to yet further embodiment, the invention provides a method forthe treatment or prevention of diseases and disorders mediated by GAGs,specifically HS-GAG. The diseases and disorders mediated by GAGs areselected from the group consisting of amyloid disorders such asAlzheimer's disease and type II diabetes; viral diseases such ashepatitis C and B, influenza, rhinovirus infections, cytomegalovirusinfections, AIDS, and respiratory syncytial virus infections; bacterialinfections and malaria; kidney diseases; cancer such as leukemia; andcoagulation disorders.

Further embodiments and the full scope of applicability of the presentinvention will become apparent from the detailed description givenhereinafter. However, it should be understood that the detaileddescription and specific examples, while indicating preferredembodiments of the invention, are given by way of illustration only,since various changes and modifications within the spirit and scope ofthe invention will become apparent to those skilled in the art from thisdetailed description.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows L-selectin binding to immobilized heparin.

FIG. 2 demonstrates inhibition of L-selectin/IgG binding to immobilizedheparin by soluble heparin.

FIG. 3 shows inhibition of L-selectin/IgG binding to immobilized heparinby anti-L-selectin antibody DREGG-55. Anti-beta-amyloid antibodies wereused as a control.

FIG. 4 shows dose-dependent inhibition of Compound no. 5 of neutrophilinfiltration in mouse peritonitis.

FIG. 5 demonstrates the anti-inflammatory properties of Compound no. 5in Paw Edema.

FIG. 6 demonstrates the anti-inflammatory properties of Compound no. 5administered intravenously in a model of Delayed Type Hypersensitivity(DTH).

FIG. 7 shows the binding of L-selectin to immobilized heparin in theabsence or presence of Compound no. 1 (100 μM).

FIG. 8 shows inhibition of L-selectin binding to immobilized bovinekidney heparan sulfate by different inhibitor Compounds.

FIG. 9 demonstrates the therapeutic effect of test Compound no. 11(TC11) administered intra-peritoneally in a mouse model of InflammatoryBowel Disease (IBD).

FIG. 10 demonstrates the therapeutic effect of test Compound no. 11(TC11) administered orally in a mouse model of Inflammatory BowelDisease (IBD)

FIG. 11 demonstrates the therapeutic effect of test Compound no. 11 in arat model of Multiple Sclerosis (Experimental AutoimmuneEncephalomyelitis).

DETAILED DESCRIPTION OF THE INVENTION DEFINITIONS

In accordance with the present invention and as used herein, thefollowing terms are defined with the following meanings, unlessexplicitly stated otherwise.

The term “compound” refers to small organic molecule having a molecularweight less than 1500 Daltons and preferably between 300 to 1200Daltons.

The term “GAG” refers to glycosaminoglycans, including heparan sulfate(HS-GAG), heparin, chondroitin sulfate, dermatan sulfate and keratansulfate. It includes the GAG chains of proteoglycans such as heparansulfate proteoglycan or chondroitin sulfate proteoglycan. It includesfragments of GAG produced chemically or enzymatically. It also includesderivatives of GAG, which may be produced by chemical or enzymatic meansas known in the art. GAG may be free or attached to a linker, support,cell or a protein. GAGs may be crude or purified from organs, tissues orcells.

The term “HS-GAG” refers to heparan sulfate glycosaminoglycan. Itincludes fragments of heparan sulfate such as those that may be producedchemically, enzymatically or during purification. It includes the HS-GAGchains of proteoglycans such as heparan sulfate proteoglycans. HS-GAGmay be free or attached to a linker, support, cell or protein, orotherwise chemically or enzymatically modified. HS-GAGs may be crude orpurified from organs, tissues or cells.

“HS-PG” refers to heparan sulfate proteoglycans.

“Heparin” is polysulfated polysaccharide, with no protein associatedwith it. As used herein, heparin refers to heparin prepared fromdifferent organs or species such as from porcine intestinal mucosa. Theinvention encompasses heparins with various molecular weights includinglow molecular weight heparins, such as commercially availableFraxiparin, and other heparin derivatives, prepared or modified bychemical or enzymatic reactions as known in the art.

“GAG Derivatives” or “ECAM Derivatives” consist of products derived fromGAGS or ECAMS, respectively, made by one or more chemical or enzymaticmodifications. The modifications are designed to modify the activity ofrelevant groups of the molecules.

“Oligosaccharide fragments” or “GAG Derived Oligosaccharides” areproducts derived from GAGs by controlled cleavage and preferablypurified after cleavage.

The terms “L-selectin/IgG” and “P-selectin/IgG” refer to a selectinchimera molecule, in which an N-terminal portion of the selectincomprising the binding domain is fused to an IgG Fc region (Aruffo etal., Cell 67:35, 1991; and Foxall et al. J. Cell Biol. 117:895, 1992).

The term “GAG specific ECAM” means an effector cell adhesion moleculeand refers to a carbohydrate-binding protein molecule involved inmediating cell adhesion, cell-cell and cell-matrix interaction andhaving a GAG binding domain. Examples of ECAMs are selectins such asL-selectin, P-selectin, integrins, fibronectin, cytokines, and the like.The term “GAG specific ECAM” also includes mutant ECAMs, proteindomains, polypeptides or peptides derived from ECAMs, chemical orenzymatic derivatives of ECAMs, and the like, so long as the mutantECAMs, protein domains, polypeptides, peptides and derivatives of ECAMsretain the capability to bind GAGs.

The term “inhibitor Compound” refers to a small organic compound thatinhibits, modulates or reverses the function of a GAG. For instance,inhibitor Compound may inhibit interaction (binding) between twomolecules: (1) a GAG, exemplified by, but not restricted to, heparin, orHS-GAG and (2) a GAG specific ECAM, exemplified by, but not restrictedto, L-selectin, P-selectin, or integrin.

The terms “inflammation”, “inflammatory diseases”, “inflammatorycondition” or “inflammatory process” are meant as physiological orpathological conditions, which are accompanied by an inflammatoryresponse. Such conditions include, but are not limited to, sepsis,ischemia-reperfusion injury, Crohn's disease, arthritis, multiplesclerosis, cardiomyopathic disease, colitis, infectious meningitis,encephalitis, acute respiratory distress syndrome, organ/tissuetransplant rejection (such as skin, kidney, heart, lung, liver, bonemarrow, cornea, pancreas, small bowel), an infection, dermatitis,stroke, traumatic brain injury, inflammatory bowel disease, andautoimmune diseases.

The term “treatment” or “treating” is intended to include theadministration of the compound of the invention to a subject forpurposes which may include prophylaxis, amelioration, prevention or cureof disorders mediated by cell adhesion or cell migration events,specifically selectin adhesion events, more specifically L-selectin andP-selectin-mediated adhesion events. Such treatment need not necessarilycompletely ameliorate the inflammatory response or other responsesrelated to the specific disorder. Further, such treatment may be used inconjunction with other traditional treatments for reducing the diseaseor disorder condition known to those of skill in the art.

The methods of the invention may be provided as a “preventive” treatmentbefore detection of, for example, an inflammatory state, so as toprevent the disorder from developing in patients at high risk for thesame, such as, for example, transplant patients.

The term “cancer” refers to various cancer-associated conditionsincluding metastasis, tumor growth, and angiogenesis. According to theinvention, cancer is exemplified by leukemias.

As used through this specification and the appended claims, the singularforms “a”, “an” and “the” include the plural unless the context clearlydictates otherwise. Thus, for example, reference to “a compound”includes mixtures of such compounds, reference to “a P-selectin”, or “anL-selectin” includes reference to respective mixtures of such molecules,reference to “the formulation” or “the method” includes one or moreformulations, methods and/or steps of the type described herein and/orwhich will become apparent to those persons skilled in the art uponreading this disclosure.

Pharmaceutical Compositions

The present invention relates to pharmaceutical compositions comprisingas an active ingredient at least one compound capable of inhibiting theinteractions of glycosaminoglycans (GAGs), particularly heparan sulfateglycosaminoglycans (HS-GAG) with effector cell adhesion molecules(ECAMs), particularly GAG-specific ECAMs, specifically L-selectin andP-selectin.

According to some aspects the present invention provides pharmaceuticalcompositions comprising as an active ingredient a compound having thegeneral formula I:

wherein:

R₁ is selected from the group consisting of H; straight or branchedalkyl of 1-6 carbon atoms; arylalkyl; substituted arylalkyl; cycloalkyl,optionally substituted with alkyl groups; alkanoyl; arylcarbonyloptionally substituted at the aryl group; cycloalkylcarbonyl;alkoxycarbonyl;

R₂ is selected from the group consisting of carboxy; cyano;aminocarbonyl; alkylaminocarbonyl; arylaminocarbonyl optionallysubstituted at the aryl group; dialkylaminocarbonyl wherein each alkylis straight or branched chain C₁-C₆ alkyl or both alkyl groups togethermay form a 3-7 membered saturated, unsaturated or aromatic monocyclic orbicyclic nitrogen containing heterocyclyl, optionally containing one ortwo additional heteroatoms; alkoxycarbonyl; alkanoyl;cycloalkylcarbonyl; arylcarbonyl optionally substituted on the arylgroup, benzothiazol-2-yl;

R₃ and R₄ are selected from the group consisting of C₁-C₆ alkyl,optionally substituted by hydroxy, alkoxy, amino or alkylamino, C₂-C₄monounsaturated alkenyl, cycloalkyl, aryl, arylmethyl, or R₃ and R₄together may form an optionally substituted 5-7 membered saturated,unsaturated or aromatic monocyclic or bicyclic nitrogen containingheterocyclyl, optionally containing one or two additional heteroatoms;

R₅, R₆, R₇ and R₈ are selected from the group consisting of H or C₁-C₆alkyl, with the proviso that when R₅, R₆, R₇ and R₈ are C₁-C₆ alkyl, R₁is hydrogen;

and pharmaceutically acceptable salts thereof; further comprising apharmaceutically acceptable diluent or carrier.

According to one embodiment, R₁ is selected from the group consisting ofmethyl, ethyl, 1-methylethyl, phenylmethyl, acetyl, ethoxycarbonyl andR₅═R₆═R₇═R₈ are hydrogen.

According to another embodiment, R₁ is hydrogen and R₅═R₆═R₇═R₈ arehydrogens or methyl groups.

According to yet another embodiment, R₁═R₅═R₆ are methyl and R₇═R₈ arehydrogens.

According to a further embodiment R₂ is selected from the groupconsisting of cyano, methoxycarbonyl, ethoxycarbonyl, aminocarbonyl,methylaminocarbonyl, dimethylaminocarbonyl, pyrrolidinylcarbonyl,piperidinylcarbonyl, morpholinylcarbonyl, benzothiazol-2-yl.

According to another embodiment, R₃ and R₄ are selected from the groupconsisting of methyl, ethyl, propyl, butyl, methoxyethyl, chlorobutyl,cyanoethyl, phenyl, cyclopentyl, cyclohexyl, phenylmethyl, allyl orcrotyl, R₃ and R₄ may be equal or different.

According to another embodiment, R₃ and R₄ form pyrrolidine, piperidine,2-methyl, 3-methyl, 4-methyl or 3,5-dimethyl piperidine,perhydroazepine, morpholine, piperazine, 4-methylpiperazine,3,4-dihydro-2(1H)-isoquinolinyl, 3,4-dihydro-1(2H)quinoline,1,3,3-trimethyl-6-azabicyclo[3.2. 1]oct-6-ane and substitutedderivatives thereof. The substituted derivatives include, but are notlimited to, piperazinyl-4-caboxylic acid ester, piperidinyl-4-carboxylicacid ester, piperidinyl-3-carboxylic acid ester.

According to one embodiment, the preferred pharmaceutical compositionsof the invention comprise as an active ingredient a compound accordingto formula I selected from:

2-[[4-[(ethylbutylamino)sulfonyl]benzoyl]amino]-3-(benzothiazol-2-yl)-6-ethyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine(Compound no. 5);

2-[[(4-(3,4-dihydro-2(1H)-isoquinolinyl)sulfonyl]benzoyl]amino]-6-(1-methylethyl)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxamide(Compound no. 1);

2-[[4-(methylphenylamino)sulfonyl]benzoyl]amino]-6-(1-methylethyl)4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxamide;

2-[[4-(3,4-dihydro-2(1H)-isoquinolinyl)sulfonyl]benzoyl]amino]-4,5,6,7-tetrahydro-5,5,7,7-tetramethylthieno[2,3-c]pyridine-3-carboxamide;

2-[[4-[(diethylamino)sulfonyl]benzoyl]amino]-3-(benzothiazol-2-yl)-6-ethyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine(Compound 11);

2-[[4-(morpholinylsulfonyl)benzoyl]amino]-3-(benzothiazol-2-yl)-6-(1-methylethyl)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine;

2-[[4-(diethylamino)sulfonyl]benzoyl]amino]-4,5,6,7-tetrahydro-5,5,7,7-tetramethylthieno[2,3-c]pyridine-3-carboxylic acid ethyl ester;

2-[[4-(3,4-dihydro-1(2H)-quinolinyl)sulfonyl]benzoyl]amino]-3-(benzothiazol-2-yl)-4,5,6,7-tetrahydro-5,5,7,7-tetramethylthieno[2,3-c]pyridine;

2-[[4-(hexahydro-1H-azepin-1-yl)sulfonyl]benzoyl]amino]-4,5,6,7-tetrahydro-5,5,7,7-tetramethylthieno[2,3-c]pyridine-3-carboxylic acid ethyl ester;

2-[[4-[[4-(methyl)-1-piperazinyl]sulfonyl]benzoyl]amino]-3-(benzothiazol-2-yl)-6-methyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine;

2-[[4-[(1,3,3-trimethyl-6-azabicyclo[3.2.1]oct-6-yl)sulfonyl]benzoyl]amino]-3-(benzothiazol-2-yl)-6-methyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine;

2-[[4-[(methylphenylamino)sulfonyl]benzoyl]amino]-3-(benzothiazol-2-yl)-6-methyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine;

2-[[4-(morpholinylsulfonyl)benzoyl]amino]-3-(benzothiazol-2-yl)-6-methyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine.

2-[[4-[(diethylamino)sulfonyl]benzoyl]amino]-3-(benzothiazol-2-yl)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-6-carboxylicacid ethyl ester;

2-[[4-[[4-(3-methyl-1-piperidinyl)]sulfonyl]benzoyl]amino]-3-(benzothiazol-2-yl)-6-methyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine;

2-[[4-[(diethylamino)sulfonyl]benzoyl]amino]-3-(benzothiazol-2-yl)-6-(phenylmethyl)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine;

2-[[4-[(diethylamino)sulfonyl]benzoyl]amino]-3-(benzothiazol-2-yl)-6-methyl4,5,6,7-tetrahydrothieno[2,3-c]pyridine;

2-[[4-[[4-(ethoxycarbonyl)-1-piperazinyl]sulfonyl]benzoyl]amino]4,5,6,7-tetrahydro-5,5,7,7-tetramethylthieno[2,3-c]pyridine-3-carboxamide;

2-[[4-[(cyclohexylmethylamino)sulfonyl]benzoyl]amino]-4,5,6,7-tetrahydro-5,5,7,7-tetramethylthieno[2,3-c]pyridine-3-carboxamide;

2-[[4-[(di-2-propenylamino)sulfonyl]benzoyl]-4,5,6,7-tetrahydro-5,5,7,7-tetramethylthieno[2,3-c]pyridine-3-carboxylicacid methyl ester;

2-[[4-[(di-2-methoxyethylamino)]sulfonyl]benzoyl]-4,5,6,7-tetrahydro-5,5,7,7-tetramethylthieno[2,3-c]pyridine-3-carboxamide;

2-[[4-[(1,3,3-trimethyl-6-azabicyclo[3.2.1.]oct-6-yl)sulfonyl]benzoyl]amino]-6methyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxamide;

2-[[4-[(diethylamio)sulfonyl]benzoyl]amino]-3-(benzothiazol-2-yl)-6-methyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine;

2-[[4-[(diethylamino)sulfonyl]benzoyl]amino]-3-(benzothiazol-2-yl)-6-(1-methylethyl)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine;

2-[[4-[(di-2-methoxyethylamino)sulfonyl]benzoyl]-amino]-3-(benzothiazol-2-yl)-6-methyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine;

2-[[4-[(methylphenylamino)sulfonyl]benzoyl]amino]-3-(benzothiazol-2-yl)-6-methyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine;

2-[[4-[[4-(ethoxycarbonyl)-1-piperazinyl]sulfonyl]benzoyl]amino]-3-(benzothiazol-2-yl)-6-methyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine;

2-[[4-[(methylbutylamino)sulfonyl]benzoyl]amino]-6-(1-methylethyl)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxylicacid ethyl ester;

2-[[4-[[4-(ethoxycarbonyl)-1-piperazinyl]sulfonyl]benzoyl]amino]-6-(1-methylethyl)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxylicacid ethyl ester;

2-[[4-(diethylamino)sulfonyl]benzoyl]amino]-4,5,6,7-tetrahydro-5,5,7,7-tetramethylthieno[2,3-c]pyridine-3-carboxamide;

2-[[4-[(methylphenylamino)sulfonyl]benzoyl]amino]-6-ethyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxylicacid methylamide;

2-[[4-[[ethyl(phenylmethyl)amino]sulfonyl]benzoyl]amino]-6-ethyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxamide;

2-[[4-[(4-methyl-1-piperazinyl)sulfonyl]benzoyl]amino]-6-ethyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxamide;

2-[[(4-(3,4-dihydro-1(2H)-quinolinyl)sulfonyl]benzoyl]amino]-6-ethyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxylicacid methylamide;

2-[[4-[(4-methyl-1-piperazinyl)sulfonyl]benzoyl]amino]-6-ethyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxylicacid methylamide;

2-[[4-[(4-methyl-1-piperazinyl)sulfonyl]benzoyl]amino]-3-(benzothiazol-2-yl)-6-ethyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine;

2-[[4-(diethylamino)sulfonyl]benzoyl]amino]-6-ethyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxylicacid N-methylamide;

2-[[4-(diethylamino)sulfonyl]benzoyl]amino]-6-ethyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxylicacid morpholinylamide.

According to another embodiment, the more preferred pharmaceuticalcompositions of the invention comprise as an active ingredient acompound of formula I selected from:

2-[[4-[(ethylbutylamino)sulfonyl]benzoyl]amino]-3-(benzothiazol-2-yl)-6-ethyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine(Compound no. 5);

2-[[4-[(diethylamino)sulfonyl]benzoyl]amino]-3-(benzothiazol-2-yl)-6-ethyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine(Compound no. 11);

2-[[4-[[ethyl(phenylmethyl)amino]sulfonyl]benzoyl]amino]-6-ethyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxamide(Compound no. 28);

2-[[4-[(4-methyl-1-piperazinyl)sulfonyl]benzoyl]amino]-6-ethyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxamide(Compound no. 29).

The present invention also provides compounds and pharmaceuticalcompositions thereof of formula2-[[4-[[ethyl(phenylmethyl)amino]sulfonyl]benzoyl]amino]-6-ethyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxamide(Compound No. 28) and2-[[4-[(4-methyl-1-piperazinyl)sulfonyl]benzoyl]amino]-6-ethyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxamide(Compound No. 29), the synthesis of the compounds is described hereinbelow in Examples 1 and 2.

Unless otherwise indicated, all chiral, diastereomeric and racemic formsof the compounds described in the present invention are also included inthe present invention; the compounds may also have asymmetric centers.Many geometric isomers of olefins, C- and N-double bonds and the likecan also be present in the compounds described herein, and all suchstable isomers are contemplated in the present invention. It will beappreciated that compounds of the present invention that containasymmetrically substituted carbon atoms may be isolated in opticallyactive or racemic forms. It is well known in the art how to prepareoptically active forms, such as by resolution of racemic forms or bysynthesis, from optically active starting materials. All chiral,diastereomeric, racemic forms and all geometric isomeric forms of astructure are intended, unless the specific stereochemistry or isomerform is specifically indicated.

When a bond to a substituent is shown to cross the bond connecting twoatoms in a ring, then such substituent may be bonded to any atom on thering. When a bond joining a substituent to another group is notspecifically shown or the atom in such other group to which the bondjoins is not specifically shown, then such substituent may form a bondwith any atom on such other group.

Combinations of substituents and/or variables are permissible only ifsuch combinations result in stable compounds. By stable compound orstable structure it is meant herein a compound that is sufficientlyrobust to survive isolation to a useful degree of purity from a reactionmixture, and formulation into an efficacious therapeutic agent.

The term “substituted”, as used herein, means that any one or morehydrogen on the designated atom is replaced with a selection from theindicated group, provided that the designated atom's normal valency isnot exceeded, and that the substitution results in a stable compound.

A “lead compound” is a compound in a selected combinatorial library, forwhich the assay has revealed significant effect relevant to a desiredcell activity to be modulated. In the present case the property is themodulation of at least one biological activity associated with a GAG orGAG-ECAM interactions.

The term “alkyl” refers to a straight or branched chain or cyclichydrocarbon having 1-12 carbon atoms. In one embodiment, the alkyl has1-10 carbons. In another embodiment, the alkyl has 1-8 carbons. Inanother embodiment, the alkyl has 1-6 carbons. In another embodiment,the alkyl has 1-4 carbons. The alkyl may be unsubstituted or substitutedby one or more substituents, i.e. substituents that do not interferewith the biological activity of the compounds. Non-limiting examples ofsuitable substituents include but are not limited to halo, hydroxy,C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl, C₁-C₁₀ alkoxy, C₇-C₁₂aralkyl, C₇-C₁₂ alkaryl, C₁-C₁₀ alkylthio, arylthio, aryloxy, arylamino,C₃-C₁₀ cycloalkyl, C₃-C₁₀ cycloalkenyl, di(C₁-C₁₀)-alkylamino, C₂-C₁₂alkoxyalkyl, C₁-C₆ alkylsulfinyl, C₁-C₁₀ alkylsulfonyl,arylsulfonyl,aryl, hydroxy, hydroxy(C₁-C₁₀)alkyl, aryloxy(C₁-C₁₀)alkyl,C₁-C₁₀ alkoxycarbonyl, aryloxycarbonyl, aryloyloxy, substituted alkoxy,fluoroalkyl, nitro, cyano, cyano(C₁-C₁₀)alkyl, C₁-C₁₀ alkanamido,aryloylamido, arylaminosulfonyl, sulfonamido, amidino, amido,alkylamido, dialkylamido, amino, alkylamino, dialkylamino, carbonyl,carbamido, carboxy, heterocyclic radical, nitroalkyl, and—(CH₂)_(m)-Z-(C₁-C₁₀alkyl), where m is 1 to 8 and z is oxygen or sulfur.The term “lower alkyl” refers to straight chain or branched alkyl groupsof 1-6 carbon atoms, such as methyl, ethyl, 1-methylethyl, propyl,butyl, isobutyl, sec-butyl, t-butyl, pentyl, isopentyl, hexyl and thelike. In one preferred embodiment, the lower alkyl is a methyl group. Inanother preferred embodiment, the lower alkyl is a methylethyl group.

The term “aryl” refers to an aromatic group having at least onecarbocyclic aromatic group, which may be unsubstituted or substituted byone or more inert substituents as defined hereinabove.

The term “heterocyclyl” or “heteroaryl” refers to a ring containing oneor more heteroatoms, for example oxygen, nitrogen, sulfur and the like,with or without unsaturation or aromatic character, optionallysubstituted with one or more inert substituents as defined hereinabove.Non-limiting examples of heterocyclic substituents are imidazole,pyrazole, pyrazine, thiazole, thiazine, oxazole, furan, dihydrofuran,tetrahydrofuran, pyridine, dihydropyridine, tetrahydropyridine,isoxazole and the like. Multiple rings may be fused, as in quinoline orbenzofuran, or unfused as in 4-phenylpyridine.

The heterocyclic moiety is a one or two ringed moiety containing one ormore heteroatoms, preferably nitrogens, which may be isolated or fused,for example and without being limited to—imidazole, pyrazole, pyrazine,pyridine, dihydropyridine, tetrahydropyridine, isoxazole, quinoline,isoquinoline and the like.

A “haloalkyl” group refers to an alkyl group as defined above, which issubstituted by one or more halogen atoms, e.g., by F, Cl, Br or I. A“hydroxyl” group refers to an OH group. An “alkenyl” group refers to agroup having at least one carbon-to-carbon double bond. An “arylalkyl”group refers to an alkyl bound to an aryl, wherein alkyl and aryl are asdefined above. An example of an arylalkyl group is a benzyl group.

As contemplated herein, the present invention further encompassesanalogs, derivatives, isomers, pharmaceutically acceptable salts andhydrates of the compounds defined by the present invention.

The term “isomer” includes, but is not limited to, optical isomers andanalogs, structural isomers and analogs, conformational isomers andanalogs, and the like. Thus, the present invention encompasses variousoptical isomers of the compounds of the present invention. It will beappreciated by those skilled in the art that the compounds of thepresent invention contain at least one chiral center. Accordingly, thesecompounds exist in, and are isolated in, optically active or racemicforms. Some compounds may also exhibit polymorphism. It is to beunderstood that the present invention encompasses any racemic, opticallyactive, polymorphic, or stereroisomeric form, or mixtures thereof. Inone embodiment, the compounds are the pure (R)-isomers. In anotherembodiment, the compounds are the pure (S)-isomers. In anotherembodiment, the compounds are a mixture of the (R) and the (S) isomers.In another embodiment, the compounds are a racemic mixture comprising anequal amount of the (R) and the (S) isomers. It is well known in the arthow to prepare optically active forms, for example, by resolution of theracemic form by recrystallization techniques, by synthesis fromoptically active starting materials, by chiral synthesis, or bychromatographic separation using a chiral stationary phase.

This invention further includes derivatives of the compounds. The term“derivatives” includes, but is not limited to, ether derivatives, acidderivatives, amide derivatives, ester derivatives, and the like. Inaddition, this invention further includes hydrates of the compoundsdescribed herein. The term “hydrate” includes but is not limited tohemihydrate, monohydrate, dihydrate, trihydrate, and the like.

The derivatives of the compounds of the present invention can also be inthe form of prodrugs. Prodrugs are considered to be any covalentlybonded carriers that release the active parent drug according to FormulaI in vivo, when such prodrug is administered to a mammalian subject.Prodrugs of the compounds of Formula I are prepared by modifyingfunctional groups present in the compounds in such a way that themodifications are cleaved, either in routine manipulation or in vivo, tothe parent compounds. Prodrugs include compounds of Formula I whereinhydroxyl, amino, sulfhydryl, or carboxyl groups are bonded to any groupthat, when administered to a mammalian subject, are cleaved to form afree hydroxyl, amino, sulfhydryl, or carboxyl group, respectively.Examples of prodrugs include, but are not limited to, acetate, formate,and benzoate derivatives of alcohol and amine functional groups in thecompounds of Formula I, and the like.

According to one embodiment, the compounds of the present inventioninhibit the interaction of GAGs with GAG specific ECAMs.

According to another embodiment, the compounds of the present inventioninhibit the interactions of HS-GAGs with selectins, specificallyL-selectin and P-selectin (see Example 5 and Table 1 herein below).

According to yet another embodiment, the compounds of the presentinvention bind directly to GAGs, specifically HS-GAG (see Example 6herein below). The compounds of the invention can thus be employed fortreatment or prevention of diseases and disorders mediated by GAGs.

According to yet another embodiment the present invention provides amethod for inhibiting cell adhesion and cell migration in vitrocomprising the step of exposing the cell to at least one compoundaccording to formula I in an amount sufficient to inhibit GAG to GAGspecific ECAM interactions (see Example 7 herein below).

The inhibitory effect of the compounds of the present invention can beevaluated by several methods in vitro. One assay for measuring GAG-ECAMbinding, exemplified herein below, detects the binding of L selectin/IgGto immobilized heparin. Another assay utilizes immobilized L-selectin,or L-selectin fused to protein domains other than IgG. The amount ofbound L-selectin is determined by an ELISA assay using, for example, amonoclonal antibody raised against L-selectin, which is conjugated tohorseradish peroxidase. FIG. 1 shows the saturation curve of theL-selectin/IgG binding to heparin. As shown in FIG. 2, soluble heparininhibited L-selectin/IgG binding to immobilized heparin. A mAb directedagainst the carbohydrate-binding domain of L-selectin (Dregg-55)inhibited L-selectin/IgG binding to heparin (FIG. 3), while anon-specific antibody such as anti-beta amyloid did not inhibit thebinding, thus providing a further confirmation of the specificity ofbinding. The Dregg-55 antibody was also shown to inhibitL-selectin-dependent adhesion in vitro, neutrophil accumulation in vitroand inflammation in vivo (Co M. S. et al, Immunotechnology 493: 253-266,1999). Thus, the experiment with Dregg-55 antibody shows that the assayaccording to the present invention is useful for discovery of compoundsinhibiting cell interaction and infiltration and having therapeuticpotential.

The biological activity of the compounds according to formula I of thepresent invention may be assayed in a variety of systems. For example, acompound can be immobilized on a solid surface and adhesion of cellsexpressing HS-GAGs can be measured. The test compounds can also betested for the ability to competitively inhibit binding between HS-GAGsand other proteins binding to HS-GAGs such as other cell adhesionmolecules, cytokines, or viral proteins. Many assay formats employradioactive or non-radioactive labeled assay components. The labelingsystems can be in a variety of forms. The label may be coupled directlyor indirectly to the desired component of the assay according to methodswell known in the art.

According to a further embodiment the compounds of the present inventioninhibit leukocyte and neutrophil infiltration in vivo (see Example 8 andFIG. 4 herein below).

The ability of compounds of the present invention to reduce leukocytemigration to sites of acute inflammation was evaluated in BALB/c miceusing a thioglycolate-induced model of peritonitis. In this animalmodel, interactions of L- and P-selectin with HS-GAGs have beenimplicated in neutrophil infiltration (Nelson, R. M., 82:3253-3258,1993; Xie, X. et al., J. Biol. Chem., 275:34818-34825, 2000).

Compounds according to formula I of the present invention were shown toefficiently inhibit leukocyte and neutrophil migration into theperitoneal cavity. The compounds were also shown to reduce lymphocytemigration, evaluated in mice using a model of Delayed TypeHypersensitivity (see Example 10 and FIG. 6 herein below; for the methodsee Lange-Asschenfeldt B. et al., Blood, 99:538-545, 2002).

Compounds of the present invention having the desired biologicalactivity may be modified as necessary to provide desired properties suchas improved pharmacological properties.

For diagnostic purposes, a wide variety of labels may be linked to thecompounds, which may provide, directly or indirectly, a detectablesignal. Thus, the compounds of the present invention may be modified ina variety of ways for a variety of end purposes while still retainingbiological activity. In addition, various reactive sites may beintroduced at the terminus for linking to particles, solid substrates,macromolecules, or the like.

Labeled compounds can be used in a variety of in vivo or in vitroapplications. A wide variety of labels may be employed, such asradionuclides (e.g., gamma-emitting radioisotopes such as technetium-99or indium-111), fluorescent agents (e.g., fluorescein), enzymes, enzymesubstrates, enzyme cofactors, enzyme inhibitors, chemiluminescentcompounds, bioluminescent compounds, and the like. Those of ordinaryskill in the art will know of other suitable labels for binding to thecompounds, or will be able to ascertain such using routineexperimentation. The binding of these labels is achieved using standardtechniques common to those of ordinary skill in the art.

For in vivo diagnostic imaging to identify, for example, sites ofinflammation, radioisotopes are typically used in accordance withwell-known techniques.

The invention includes pharmaceutically acceptable salts of thecompounds of the present invention. Pharmaceutically acceptable saltscan be prepared by treatment with inorganic bases, for example, sodiumhydroxide or inorganic/organic acids such as hydrochloric acid, citricacids and the like.

The term “pharmaceutically acceptable salts” refers to salts preparedfrom pharmaceutically acceptable non-toxic bases or acids includinginorganic or organic bases and inorganic or organic acids. Salts derivedfrom inorganic bases include aluminum, ammonium, calcium, copper,ferric, ferrous, lithium, magnesium, manganic salts, manganous,potassium, sodium, zinc, and the like. Particularly preferred are theammonium, calcium, magnesium, potassium, and sodium salts. Salts derivedfrom pharmaceutically acceptable organic non-toxic bases include saltsof primary, secondary, and tertiary amines, substituted amines includingnaturally occurring substituted amines, cyclic amines, and basic ionexchange resins, such as arginine, betaine, caffeine, choline,N,N′-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol,2-dimethylaminoethanol, ethanolamine, ethylenediamine,N-ethyl-morpholine, N-ethylpiperidine, glucamine, glucosamine,histidine, hydrabamine, isopropylamine, lysine, methylglucamine,morpholine, piperazine, piperidine, polyamine resins, procaine, purines,theobromine, triethylamine, trimethylamine, tripropylamine,tromethamine, and the like.

When the compound of the present invention is basic, salts may beprepared from pharmaceutically acceptable non-toxic acids, includinginorganic and organic acids. Such acids include acetic, benzenesulfonic,benzoic, camphorsulfonic, citric, ethanesulfonic, fumaric, gluconic,glutamic, hydrobromic, hydrochloric, isethionic, lactic, maleic, malic,mandelic, methanesulfonic, mucic, nitric, pamoic, pantothenic,phosphoric, succinic, sulfuric, tartaric, p-toluenesulfonic acid, andthe like. Particularly preferred are citric, hydrobromic, hydrochloric,maleic, phosphoric, sulfuric, and tartaric acids.

It is to be understood that, as used herein, references to the compoundsaccording to formula I of the present invention are meant to alsoinclude the pharmaceutically acceptable salts thereof.

Pharmaceutical Formulations

The pharmaceutical compositions of the present invention can beformulated for administration by a variety of routes including oral,rectal, transdermal, subcutaneous, intravenous, intramuscular, andintranasal. Such compositions are prepared in a manner well known in thepharmaceutical art and comprise as an active ingredient at least onecompound according to formula I and derivatives thereof as describedherein above, further comprising an excipient or a carrier. During thepreparation of the pharmaceutical compositions according to the presentinvention the active ingredient is usually mixed with an excipient,diluted by an excipient or enclosed within such a carrier which can bein the form of a capsule, sachet, paper or other container. When theexcipient serves as a diluent, it can be a solid, semi-solid, or liquidmaterial, which acts as a vehicle, carrier or medium for the activeingredient. Thus, the compositions can be in the form of tablets, pills,powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions,solutions, syrups, aerosols (as a solid or in a liquid medium),ointments containing, for example, up to 10% by weight of the activecompound, soft and hard gelatin capsules, suppositories, sterileinjectable solutions, and sterile packaged powders.

In preparing a formulation, it may be necessary to mill the activeingredient to provide the appropriate particle size prior to combiningwith the other ingredients. If the active compound is substantiallyinsoluble, it ordinarily is milled to a particle size of less than 200mesh. If the active ingredient is substantially water soluble, theparticle size is normally adjusted by milling to provide a substantiallyuniform distribution in the formulation, e.g. about 40 mesh.

Some examples of suitable excipients include lactose, dextrose, sucrose,sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates,tragacanth, gelatin, calcium silicate, microcrystalline cellulose,polyvinylpyrrolidone, cellulose, water, syrup, and methylcellulose. Theformulations can additionally include lubricating agents such as talc,magnesium stearate, and mineral oil; wetting agents; emulsifying andsuspending agents; preserving agents such as methyl- andpropylhydroxybenzoates; sweetening agents; and flavoring agents. Thecompositions of the invention can be formulated so as to provide quick,sustained or delayed release of the active ingredient afteradministration to the patient by employing procedures known in the art.

The compositions are preferably formulated in a unit dosage form, eachdosage containing from about 0.1 to about 500 mg. The term “unit dosageforms” refers to physically discrete units suitable as unitary dosagesfor human subjects and other mammals, each unit containing apredetermined quantity of the active compound calculated to produce thedesired therapeutic effect, in association with a suitablepharmaceutical excipient.

The active ingredient is effective over a wide dosage range and isgenerally administered in a therapeutically effective amount. It will beunderstood, however, that the amount of the compound actuallyadministered will be determined by a physician, in the light of therelevant circumstances, including the condition to be treated, thechosen route of administration, the actual compound administered, theage, weight, and response of the individual patient, the severity of thepatient's symptoms, and the like.

For preparing solid compositions such as tablets, the principal activeingredient is mixed with a pharmaceutical excipient to form a solidpreformulation composition containing a homogeneous mixture of acompound of the present invention. When referring to thesepreformulation compositions as homogeneous, it is meant that the activeingredient is dispersed evenly throughout the composition so that thecomposition may be readily subdivided into equally effective unit dosageforms such as tablets, pills and capsules. This solid preformulation isthen subdivided into unit dosage forms of the type described abovecontaining from, for example, 0.1 to about 500 mg of the activeingredient of the present invention.

The tablets or pills of the present invention may be coated or otherwisecompounded to provide a dosage form affording the advantage of prolongedaction. For example, the tablet or pill can comprise an inner dosage andan outer dosage component, the latter being in the form of an envelopeover the former. The two components can be separated by an entericlayer, which serves to resist disintegration in the stomach and permitthe inner component to pass intact into the duodenum or to be delayed inrelease. A variety of materials can be used for such enteric layers orcoatings; such materials include a number of polymeric acids andmixtures of polymeric acids with materials such as shellac, cetylalcohol, and cellulose acetate.

The liquid forms in which the compositions of the present invention maybe incorporated, for administration orally or by injection, includeaqueous solutions, suitably flavored syrups, aqueous or oil suspensions,and flavored emulsions with edible oils such as cottonseed oil, sesameoil, coconut oil, or peanut oil, as well as elixirs and similarpharmaceutical vehicles.

Compositions for inhalation or insulation include solutions andsuspensions in pharmaceutically acceptable aqueous or organic solvents,or mixtures thereof, and powders. The liquid or solid compositions maycontain suitable pharmaceutically acceptable excipients as describedabove. Preferably the compositions are administered by the oral or nasalrespiratory route for local or systemic effect. Compositions inpreferably pharmaceutically acceptable solvents may be nebulized by useof inert gases. Nebulized solutions may be breathed directly from thenebulizing device or the nebulizing device may be attached to a facemasks tent, or intermittent positive pressure breathing machine.Solution, suspension, or powder compositions may be administered,preferably orally or nasally, from devices that deliver the formulationin an appropriate manner.

Another preferred formulation employed in the methods of the presentinvention employs transdermal delivery devices (“patches”). Suchtransdermal patches may be used to provide continuous or discontinuousinfusion of the compounds of the present invention in controlledamounts. The construction and use of transdermal patches for thedelivery of pharmaceutical agents is well known in the art. See, e.g.,U.S. Pat. No. 5,023,252 incorporated herein by reference as if fully setforth. Such patches may be constructed for continuous, pulsatile, or ondemand delivery of pharmaceutical agents.

Direct or indirect placement techniques may be used when it is desirableor necessary to introduce the pharmaceutical composition to the brain.Direct techniques usually involve placement of a drug delivery catheterinto the host's ventricular system to bypass the blood-brain barrier.One such implantable delivery system used for the transport ofbiological factors to specific anatomical regions of the body isdescribed in U.S. Pat. No. 5,011,472 incorporated herein by reference asif fully set forth. Indirect techniques, which are generally preferred,usually involve formulating the compositions to provide for druglatentiation by the conversion of hydrophilic drugs into lipid-solubledrugs. Latentiation is generally achieved through blocking of thehydroxy, carbonyl, sulfate, and primary amine groups present on the drugto render the drug more lipid soluble and amenable to transportationacross the blood-brain barrier. Alternatively, the delivery ofhydrophilic drugs may be enhanced by intra-arterial infusion ofhypertonic solutions, which can transiently open the blood-brainbarrier.

Therapeutic Use

The present invention provides small organic compounds that inhibitcell-matrix and cell-cell interaction, thus inhibiting a cascade ofevents that lead to the development of certain diseases and disorders.

According to some aspects, the present invention provides a method forthe treatment or prevention of diseases and disorders related to celladhesion and cell migration mediated by GAG-ECAM interactions,comprising the step of administering to a subject in need thereof atherapeutically effective amount of a pharmaceutical compositioncomprising a compound having the general formula I:

wherein:

R₁ is selected from the group consisting of H; straight or branchedalkyl of 1-6 carbon atoms; arylalkyl; substituted arylalkyl; cycloalkyl,optionally substituted with alkyl groups; alkanoyl; arylcarbonyloptionally substituted at the aryl group; cycloalkylcarbonyl;alkoxycarbonyl;

R₂ is selected from the group consisting of carboxy; cyano;aminocarbonyl; alkylaminocarbonyl; arylaminocarbonyl optionallysubstituted at the aryl group; dialkylaminocarbonyl wherein each alkylis straight or branched chain C₁-C₆ alkyl or both alkyl groups togethermay form a 3-7 membered saturated, unsaturated or aromatic monocyclic orbicyclic nitrogen containing heterocyclyl, optionally containing one ortwo additional heteroatoms; alkoxycarbonyl; alkanoyl;cycloalkylcarbonyl; arylcarbonyl optionally substituted on the arylgroup, benzothiazol-2-yl;

R₃ and R₄ are selected from the group consisting of C₁-C₆ alkyl,optionally substituted by hydroxy, alkoxy, amino or alkylamino, C₂-C₄monounsaturated alkenyl, cycloalkyl, aryl, arylmethyl, or R₃ and R₄together may form an optionally substituted 5-7 membered saturated,unsaturated or aromatic monocyclic or bicyclic nitrogen containingheterocyclyl, optionally containing one or two additional heteroatoms;

R₅, R₆, R₇ and R₈ are selected from the group consisting of H or C₁-C₆alkyl, with the proviso that when R₅, R₆, R₇ and R₈ are C₁-C₆ alkyl, R₁is hydrogen;

and pharmaceutically acceptable salts thereof; further comprising apharmaceutically acceptable diluent or carrier.

The preferred pharmaceutical compositions for the treatment andprevention of diseases and disorders related to cell adhesion and cellmigration mediated by GAG-ECAM interactions are listed herein above.

According to one embodiment, the pharmaceutical compositions accordingto the present invention are used for the treatment of diseases ordisorders related to GAG-ECAM interactions wherein the GAGs are selectedfrom the group consisting of heparan sulfate (HS-GAG), heparin,chondroitin sulfate, dermatan sulfate, keratan sulfate, and derivativesand fragments thereof.

According to one currently preferred embodiment, the pharmaceuticalcompositions according to the present invention are used for thetreatment of diseases or disorders related to GAG-ECAM interactionswherein the GAG is HS-GAG.

According to yet another embodiment, the pharmaceutical compositionsaccording to the present invention are used for the treatment ofdiseases or disorders related to GAG-ECAM interactions wherein the GAGspecific ECAMs are selected from the group consisting of selectins,integrins, fibronectin, and cytokines.

According to one currently preferred embodiment, the pharmaceuticalcompositions according to the present invention are used for thetreatment of diseases or disorders related to GAG-ECAM interactionswherein the GAG specific ECAMs are selected from the group consisting ofL-selectin and P-selectin.

Anti cell adhesion and anti-cell migration therapy has proven to behighly effective in the treatment of number of diseases and disordersincluding inflammatory processes, autoimmune processes, cancer and tumormetastasis, and platelet-mediated pathologies.

A number of inflammatory disorders associated with L- and P-selectin orinvolve selectin-mediated leukocyte flow along the blood stream may betreated with the pharmaceutical compositions of the invention. Treatabledisorders include, but are not limited to, organ or tissuetransplantation rejection (e.g., allograft rejection or autologous bonemarrow transplantation), atherosclerosis, retinitis, cancer metastases,rheumatoid arthritis, acute leukocyte-mediated lung injury (e.g., adultrespiratory distress syndrome), asthma, allergic rhinitis, allergicconjunctivitis, inflammatory lung diseases, restenosis, nephritis, acuteand chronic inflammation, atopic dermatitis, psoriasis, contact dermalhypersensitivity, myocardial ischemia, and inflammatory bowel disease.In preferred embodiments the pharmaceutical compositions are used totreat inflammatory disorders associated with neutrophil infiltration,such as ischemia-reperfusion injury, acute pancreatitis, septic shock,uveitis, rheumatoid arthritis, and inflammatory bowel disease.

Reperfusion injury is a major problem in clinical cardiology.Therapeutic agents that reduce leukocyte adherence in ischemicmyocardium can significantly enhance the therapeutic efficacy ofthrombolytic agents. Thrombolytic therapy with agents such as tissueplasminogen activator or streptokinase can relieve coronary arteryobstruction in many patients with severe myocardial ischemia prior toirreversible myocardial cell death. However, many such patients stillsuffer myocardial necrosis despite restoration of blood flow. This“reperfusion injury” is known to be associated with adherence ofleukocytes to vascular endothelium in the ischemic zone, presumably inpart because of activation of platelets and endothelium by thrombin andcytokines that makes them adhesive for leukocytes (Romson et al.,Circulation 67:1016-1023, 1983). These adherent leukocytes can migratethrough the endothelium and ischemic myocardium just as it is beingrescued by restoration of blood flow.

Inflammatory bowel disease is a collective term for two similar diseasesreferred to as Crohn's disease and ulcerative colitis. Crohn's diseaseis an idiopathic, chronic ulceroconstrictive inflammatory diseasecharacterized by sharply delimited and typically transmural involvementof all layers of the bowel wall by a granulomatous inflammatoryreaction. Any segment of the gastrointestinal tract, from the mouth tothe anus, may be involved, although the disease most commonly affectsthe terminal ileum and/or colon. Ulcerative colitis is an inflammatoryresponse limited largely to the colonic mucosa and submucosa.Lymphocytes and macrophages are numerous in lesions of inflammatorybowel disease and may contribute to inflammatory injury.

According to yet another embodiment, the invention provides a method forthe prevention or treatment of inflammatory bowel disease comprising thestep of administering to a subject in need thereof a therapeuticallyeffective amount of a pharmaceutical composition comprising as an activeingredient a thieno[2,3-c]pyridine compound of the formula2-[[4-[(diethylamino)sulfonyl]benzoyl]amino]-3-(benzothiazol-2-yl)-6-ethyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine(Compound no. 11) and a pharmaceutically acceptable salt; furthercomprising a pharmaceutically acceptable carrier. As described inExample 8 herein below, Compound no. 5 and Compound no. 11 inhibitedleukocyte and neutrophil infiltration into mouse peritoneum. Leukocyteand neutrophil infiltration is considered a hallmark of inflammatorybowel disease. In addition, Example 11 herein below demonstrated thatCompound no. 11 significantly inhibited colonic damage in a mouse modelof inflammatory bowel disease. As shown in FIGS. 9 and 10, thetherapeutic effect of Compound no. 11 was dose-dependent andstatistically significant both after intra-peritoneal as well as oraladministration.

Asthma is a disease characterized by increased responsiveness of thetracheobronchial tree to various stimuli potentiating paroxysmalconstriction of the bronchial airways. The stimuli cause release ofvarious mediators of inflammation that recruit basophils, eosinophilsand neutrophils, which cause inflammatory injury.

Rheumatoid arthritis is a chronic, relapsing inflammatory disease thatprimarily causes impairment and destruction of joints. Rheumatoidarthritis usually first affects the small joints of the hands and feetbut then may involve the wrists, elbows, ankles and knees. The arthritisresults from interaction of synovial cells with leukocytes thatinfiltrate from the circulation into the synovial lining of the joints.

Atherosclerosis is a disease of arteries. The basic lesion, theatheroma, consists of a raised focal plaque within the intima, having acore of lipid and a covering fibrous cap. Atheromas compromise arterialblood flow and weaken affected arteries. Myocardial and cerebralinfarcts are a major consequence of this disease. Macrophages andleukocytes are recruited to atheromas and contribute to inflammatoryinjury.

The pharmaceutical compositions of the present invention can be furtherused in the treatment of organ or graft rejection. Over recent yearsthere has been a considerable improvement in the efficiency of surgicaltechniques for transplanting tissues and organs such as skin, kidney,liver, heart, lung, pancreas and bone marrow. Perhaps the principaloutstanding problem is the lack of satisfactory agents for inducingimmnunotolerance in the recipient to the transplanted allograft ororgan. When allogeneic cells or organs are transplanted into a host, thehost immune system is likely to mount an immune response to foreignantigens in the transplant (host-versus-graft disease) leading todestruction of the transplanted tissue. CD8⁺ cells, CD4 cells andmonocytes are all involved in the rejection of transplant tissues.Compounds of this invention, which inhibit selectins are useful, interalia, to block alloantigen-induced immune responses thereby preventingsuch cells from participating in the destruction of the transplantedtissue or organ (see, e.g., Georczynski et al., Immunology 87:573-580,1996).

A related use of the pharmaceutical compositions according to thepresent invention is in modulating the immune response involved in“graft versus host” disease (GVHD). GVHD is a potentially fatal diseasethat occurs when immunologically competent cells are transferred to anallogeneic recipient. In this situation, the donor's immunocompetentcells may attack tissues in the recipient. Tissues of the skin, gutepithelia and liver are frequent targets and may be destroyed during thecourse of GVHD. The disease presents an especially severe problem whenimmune tissue is being transplanted, such as in bone marrowtransplantation; but less severe GVHD has also been reported in othercases as well, including heart and liver transplants. The compounds ofthe present invention that modulate donor cell homing pattern mediatedby L-selectin are useful for treatment of GVHD (Li, B., Eur J Immunol31:617-24, 2001).

Further use of the pharmaceutical compositions according to the presentinvention is for the treatment of cancer and metastasis. As described inExample 13 herein below, Compound no. 1 inhibited the growth of severaltumor cell lines. For certain cancers to spread throughout a patient'sbody, a process of cell-cell adhesion, or metatasis, must take place.Specifically, cancer cells must migrate from their site of origin andgain access to a blood vessel to facilitate colonization at distantsites. A critical aspect of this process is adhesion of cancer cells (toplatelets and to endothelial cells that line the blood vessel wall) astep prior to migrating into surrounding tissue. This process can beinterrupted by the administration of compounds of the invention, whichgenerally aid in blocking cell-cell adhesion. In particular, P-selectinmediated processes have been implicated in metastasis (Varki, A. andVarki, N. M., Braz J Med Biol Res. 34:711-7, 2001)

Also embodied is the use of the pharmaceutical compositions according tothe present invention in the treatment of leukemia, such as AcuteMyeloid Leukemia, which involves extravasation of leukemic cells andtumor formation. As described in Example 14, Compound no. 1 inhibitedleukemia cell growth. Also embodied in the present invention are methodsuseful for the treatment (including prevention) of angiogenic disorders.The term “angiogenic disorders” as used herein includes conditionsinvolving abnormal neovascularization, such as tumor metastasis andocular neovascularization, including, for example, diabetic retinopathy,and neovascular glaucoma.

A further use of the pharmaceutical compositions according to thepresent invention is in treating multiple sclerosis. Multiple sclerosisis a progressive neurological autoimmune disease that is thought to bethe result of a specific autoimmune reaction in which certain leukocytesinitiate the destruction of myelin, the insulating sheath covering nervefibers. Murine monoclonal antibodies directed against L-selectin havebeen shown to suppress experimental autoimmune encephalomyelitis (EAE),an animal model of multiple sclerosis (Archelos, J. J., J. Neurol. Sci.,159:127-34, 1998).

The present invention also provides a method for the prevention ortreatment of multiple sclerosis comprising the step of administering toa subject in need thereof a therapeutically effective amount of apharmaceutical composition comprising as an active ingredient thethieno[2,3-c]pyridine compound2-[[4-[(diethylamino)sulfonyl]benzoyl]amino]-3-(benzothiazol-2-yl)-6-ethyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine(Compound no. 11) and a pharmaceutically acceptable carrier. As shown inFIG. 11, Compound no. 11 inhibited symptoms of EAE at two concentrationstested.

It has also been found that compounds according to formula I of thepresent invention directly bind to HS-GAGs and may therefore be usefulfor treatment of disease conditions mediated by HS-GAGs. HS-GAG mediatedconditions include those mediated by cell-cell, cell-virus, cell-matrixand cell-protein interactions. Examples of HS-GAG mediated conditionsinclude virus attachment to cell, cell adhesion, platelet aggregation,lymphocyte adhesion and migration, and amyloid fibril formation.

According to another embodiment, the-pharmaceutical compositionsaccording to the present invention are therefore also useful fortreatment (or prevention) of viral disorders such as hepatitis C and B,cytomegalovirus infection, respiratory syncytial virus infection, andAIDS.

According to yet another embodiment, the pharmaceutical compositions ofthe present invention are useful for the treatment or prevention ofatherosclerosis, amyloid disorders including Alzheimer's disease andtype II diabetes (Non-insulin Dependent Diabetes Mellitus), inflammatoryand immune disorders, cancer, bone degradation, osteoporosis,osteoarthritis, tumor metastasis, and kidney disease includingglomerulonephritis.

According to yet another embodiment, the pharmaceutical compositions ofthe present invention are used for the treatment or prevention ofcoagulation disorders. The compounds of the present invention may beuseful for counteracting the actions of heparin and other anticoagulantglycosaminoglycans on thrombin and Factor Xa activity, and nay affectother coagulation proteins as well. Heparin is used routinely foranticoagulation. The interactions of exogenously administered heparinwith the proteins of the coagulation and fibrinolytic pathways have beensummarized in detail (see for example Van Kuppevelt, T. H., et al., JBiol Chem, 273: 12960-12966, 1998). It is often necessary to reverse theeffects of heparin when anticoagulation has reached a stage at whichhemorrhage becomes a threat, notably after the routine use of heparinfor anticoagulation during cardiopulmonary bypass, and in patients whodevelop an endogenous heparin-like coagulation inhibitor. Currently, theonly FDA-approved heparin antidote available is Protamine. Protamine isa mixture of basic proteins from fish sperm nuclei that contains a highconcentration of the amino acid arginine. When injected into a personwho has been treated with heparin, Protamine complexes rapidly to theheparin, thereby neutralizing its activity. Although Protamine iseffective in humans against unfractionated heparin, it is not effectiveagainst low molecular weight heparins or against the non-heparinglycosaminoglycan anticoagulant Orgaran®, i.e., a mixture of chondroitinsulfate/heparan sulfate/dermatan sulfate. Protamine also has numerousside effects including pulmonary hypotension that are difficult tocontrol and provide significant health risks to the patient. Also, sinceProtamine is obtained from a natural source, it is a poorly-defined andpotentially variable product, dosage determination can be problematic.Well-defined heparin- or other GAG-binding compounds could be ofconsiderable utility for reversing overdose of these specificanticoagulant preparations. Carson and co-workers (Munro, M. S., et al.,Trans Am Soc Artif Intern Organs, 27: 499-503,1983) have identified aheparin-binding peptide from an epithelial/endothelial cell surfaceprotein that has some ability to neutralize heparin effects on thrombingeneration, but optimal effects were found only at high peptideconcentrations and low heparin and low thrombin concentrations. Thesmall organic compounds of the invention would be substantiallypreferable over these peptides as they are more stable and costeffective. The compounds of the invention can thus be useful inneutralization of unfractionated heparin, low molecular weight heparin,or Orgaran.

Multiple interactions between the proteins of the coagulation andfibrinolysis pathways and endothelial cell surface PGs are normallybalanced on the surface of the endothelial cells in order to create anon-thrombotic state. Heparin-binding compounds of the invention couldbehave similarly to platelet factor 4 (PF4) in that they could bind toheparin, reduce the anticoagulant activity, which occurs on the surfaceof endothelial cells, and thereby enable a clot to form.

Additional possible use for the compounds of the present invention is toblock the uptake and clearance of heparin by blocking heparin receptorsin tissues without binding to circulating heparin, and thus to prolongthe half-life of heparin in the circulation. Use of the compounds of theinvention would reduce the frequency of administration of heparin, aswell as the amount needed. This could be especially useful forhome-based therapy with low molecular weight heparin, which isadministered by subcutaneous injection and is becoming the standardpost-hospitalization anticoagulation treatment.

It is to be understood that while the compounds according to formula Iof the present invention were selected for their capacity to inhibitbinding of certain selectins to HS-GAGs, and that this propertycontributes to their medical activity, it cannot, however, be excludedthat the compounds are also exerting their favorable medical effects,either in parallel or in tandem, through additional mechanisms ofaction. Thus, the skilled practitioner of this art will appreciate thatone aspect of the present invention is the description of novelpharmaceutical compositions, and that Applicants intend not to be boundby a particular mechanism of action that may account for theirprophylactic or therapeutic effects.

The principles of the invention, providing novel compounds capable ofinhibiting GAGs-ECAMs interactions, their pharmaceutical compositionsand use thereof according to the present invention, may be betterunderstood with reference to the following non-limiting examples.

EXAMPLES

Example 1

General Synthesis of Compounds of Formula I

Compounds of Formula I are synthesized according to scheme of thereaction described by Noravyan et al. (A. S. Noravyan et al.,Khim.-Farm. Zh., 14, 37-40, 1980). A compound according to formula Aherein below (R₁ and R₂ are defined as in formula I) is reacted with anacid chloride of the compound according to formula B herein below (whereR₃ and R₄ are defined as in formula I) in dry benzene under reflux, inthe presence of triethylamine. The precipitated crystals oftriethylamine hydrochloride are filtered off and the filtrate isevaporated under slight vacuum. Work up of the residue affords thetarget compounds of formula I in 60-80% yield.

Example 2 Synthesis of Compound No. 29

The synthesis of the following compound:

2-[[4-[(4-methyl-1-piperazinyl)sulfonyl]benzoyl]amino]-6-ethyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxamide(Compound no. 29) was performed as follows:

To a solution of 2.25 g (10 mmol)2-amino-6-ethyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxamide indry toluene 2.0 g (20 mmol) triethylamine and 3.6 g (12 mmol)p-[(4-methyl-1-piperazinyl)sulfonyl]benzoyl chloride in 30 ml of drytoluene were added. The reaction mixture was refluxed for 3 h andcooled. The precipitated crystals of triethylamine hydrochlorid werefiltered off. The filtrate was evaporated in vacuum and the residuecrystallized on addition of ethyl acetate. The yellow crystals werewashed with ethyl acetate and dried. The target compound was obtained in72% yield, melting point 232° C.

¹H NMR (DMSO-d₆) δ (ppm): 7.80-8.12 (4H,m, benzene), 3,8 (2H, m, CH₂),2.82 (8H, t, CH₂-piperazine),2.28-2.41 (6H, m, 3CH₂), 1.14 (3H, t, CH₃),0.90 (3H, t, NCH₃).

Example 3

Synthesis of Compound No. 28

The synthesis of the following compound:

2-[[4-[[ethyl(phenylmethyl)amino]sulfonyl]benzoyl]amino]-6-ethyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxamide(Compound no. 28) was performed as follows:

To a solution of 2.25 g (10 mmol)2-amino-6-ethyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxamide indry toluene 2.0 g (20 mmol) triethylamine and 4.0 g (12mmol)p-[[ethyl(phenylmethyl)amino]sulfonyl]benzoyl chloride in 30 ml of drytoluene were added. The reaction mixture was refluxed for 3 h andcooled. The precipitated triethylamine hydrochloride was filtered off.The filtrate was evaporated in vacuum and the residue crystallized onaddition of ethyl acetate. The yellow crystals were washed with ethylacetate and dried. The target compound was obtained in 65% yield,melting point 246° C.

¹H NMR (DMSO-d₆) δ (ppm): 8.03-8.10 (4H, m, benzene), 7.28-7.33 (5H, m,benzene), 4.36 (2H, s, NCH₂Ph), 3.58 (2H, m, CH₂), 3.18 (2H, m, CH₂),2.50-2.90 (6H, m, CH₂), 1.34 (3H, t, CH₃), 0.88 (3H, t, CH₃).

Example 4 Pharmaceutical Compositions

The pharmaceutical compositions of the present invention are illustratedby the following formulation examples:

Formulation Example 1

Hard gelatin capsules containing the following ingredients are prepared:Ingredient Quantity (mg/capsule) Active Ingredient 30.0 Starch 305.0Magnesium stearate 5.0

The above ingredients are mixed and filled into hard gelatin capsules in340 mg quantities.

Formulation Example 2

A tablet formula is prepared using the ingredients below: IngredientQuantity (mg/tablet) Active Ingredient 25.0 Cellulose, microcrystalline200.0 Colloidal silicon dioxide 10.0 Stearic acid 5.0

The components are blended and compressed to form tablets, each weighing240 mg.

Formulation Example 3

A dry powder inhaler formulation is prepared containing the followingcomponents: Ingredient Weight % Active Ingredient 5.0 Lactose 95.0

The active ingredient is mixed with the lactose and the mixture is addedto a dry powder inhaling-appliance.

Formulation Example 4

Tablets, each containing 30 mg of active ingredient, are prepared asfollows: Ingredient Quantity (mg/tablet) Active Ingredient 30.0 Starch45.0 Microcrystalline cellulose 35.0 Polyvinylpyrrolidone 4.0 (as 10%solution in water) Sodium carboxymethyl starch 4.50 Magnesium stearate0.5 Talc 1.0

The active ingredient, starch and cellulose are passed through a No. 20mesh U.S. sieve and mixed thoroughly. The solution ofpolyvinylpyrrolidone is mixed with the resultant powders, which are thenpassed through a 16 mesh U.S. sieve. The granules so produced are driedat 50° C. to 60° C. and passed through a 16 mesh U.S. sieve. The sodiumcarboxymethyl starch, magnesium stearate, and talc, previously passedthrough a No. 30 mesh U.S. sieve, are then added to the granules which,after mixing, are compressed on a tablet machine to yield tablets eachweighing 120 mg.

Formulation Example 5

Capsules, each containing 40 mg of the active ingredient are made asfollows: Ingredient Quantity (mg/capsule) Active Ingredient 40.0 Starch109.0 Magnesium stearate 1.0

The active ingredient, starch, and magnesium stearate are blended,passed through a No. 20 mesh U.S. sieve, and filled into hard gelatincapsules in 150 mg quantities.

Formulation Example 6

Suppositories, each containing 25 mg of active ingredient are made asfollows: Ingredient Quantity (mg) Active Ingredient 25.0 Saturated fattyacid glycerides 2000.0

The active ingredient is passed through a No. 60 mesh U.S. sieve andsuspended in the saturated fatty acid glycerides previously melted usingthe minimum heat necessary. The mixture is then poured into asuppository mold of nominal 2.0 g capacity and allowed to cool.

Formulation Example 7

Suspensions, each containing 50 mg of an active ingredient per 5.0 mldose are made as follows: Ingredient Quantity (mg) Active Ingredient50.0 mg Xanthan gum 4.0 mg Sodium carboxymethyl cellulose (11%) 50.0 mgMicrocrystalline cellulose (89%) Sucrose 1.75 g Sodium benzoate 10.0 mgFlavor and Color q.v. mg Purified water to 5.0 ml

The active ingredient, sucrose and xanthan gum are blended, passedthrough a No. 10 mesh U.S. sieve, and then mixed with a previously madesolution of the microcrystalline cellulose and sodium carboxymethylcellulose in water. The sodium benzoate, flavor, and color are dilutedwith some of the water and added with stirring. Sufficient water is thenadded to produce the required volume.

Formulation Example 8

Capsules, each containing 15 mg of an active ingredient, are made asfollows: Ingredient Quantity (mg/capsule) Active Ingredient 15.0 Starch407.0 Magnesium stearate 3.0

The active ingredient, cellulose, starch, and magnesium stearate areblended, passed through a No. 20 mesh U.S. sieve, and filled into hardgelatin capsules in 425 mg quantities.

Formulation Example 9

An intravenous formulation may be prepared as follows: IngredientQuantity Active Ingredient 250.0 mg Isotonic saline 1000 ml

A topical formulation may be prepared as follows: Ingredient QuantityActive Ingredient 1-10 g Emulsifying Wax 30 g Liquid Paraffin 20 g WhiteSoft Paraffin to 100 g

The white soft paraffin is heated until molten. The liquid paraffin andemulsifying wax are incorporated and stirred until dissolved. The activeingredient is added and stirring is continued until dispersed. Themixture is then cooled until solidified.

Example 5 In vitro Assay for Determining Inhibition of L-Selectin(P-Selectin) Binding to HS-GAGs by Inhibitor Compounds According toFormula I

An in vitro assay was used to assess the ability of test compoundsaccording to formula I to inhibit the interactions of L-selectin withHS-GAGs. The assay was suitable for determining the concentrationrequired for 50% inhibition (IC-50) for each specific compound. In theassay, the GAG used was heparin. Thus, porcine intestinal mucosa heparinconjugated to Bovine Serum Albumin (Heparin-BSA; Sigma Cat. No. H0403)at 5 mg/mi in Phosphate Buffered Saline (PBS; pH 6.5) was added to a 96well polystyrene ELISA plate (NUNC Cat. No. 442404; 0.1 ml per well) andincubated Over Night (ON) at 4° C. Following the incubation, the platewas washed thoroughly, by immersion, with de-ionized water and PBS (pH6.5). The ELISA plate was then blocked with BSA (ICN Cat. No.160069, 3%,200 μl per well) for 1 hour at Room Temperature (RT). Followingblocking, the plate was washed with de-ionized water, and then with PBS(pH 6.5) containing Tween 20 (Sigma Cat. No. P-1379, 0.05%). Compoundswere synthesized or purchased from ChemDiv Labs (San Diego, Calif.),dissolved in DMSO, diluted in PBS and added to the wells at variousconcentrations in the range of 0.01 to 300 μM. Recombinant HumanL-Selectin/IgG (Research and Development Systems Cat. No.728-LS)dissolved in PBS supplemented with BSA (0.1%) and calcium chloride (1mM) was added to the ELISA plate (100 μl per well) and incubated for 60minutes at RT with shaking. Following incubation, the plate was washedwith de-ionized water and three times with PBS (pH 6.5) containing Tween20. Anti-Human IgG Peroxidase Conjugate (1:5000; Sigma Product No.A8667) diluted in PBS supplemented with BSA (0.1%) and calcium chloride(1 mM) was added to the ELISA plate (100 μl per well) and incubated for30 minutes at RT with shaking. The plate was then washed with de-ionizedwater and three times with PBS (pH 6.5) containing Tween 20. Theperoxidase substrate cromogen, TMB (Dako Cat. No. S1 599) was added (100μl per well) to the ELISA plate and incubated at room temperature. After15 minutes ELISA Stop Solution (hydrochloric acid 1N, sulfuric acid 3N)was added (200 μl per well) to stop the peroxidase catalyzedcolorimetric reaction. The Optical Density (OD) of the samples wasmeasured at 450 nm using an ELISA plate reader (Dynatech MR5000). Datawere analyzed with Graphpad Prism software and IC-50 values wereestablished. The P-selectin assay was carried out in a similar fashion,except that Recombinant Human P-Selectin/IgG (Research and DevelopmentSystems Cat.No.137-PS) was used.

It was established that compounds of Formula I had inhibitory activityin the above assays. Examples of inhibitor Compounds are given inTable 1. TABLE 1 Inhibition of L-Selectin Binding to Heparin by SelectedCompounds Compound % Inhibition IC-50 No. Structure at 50 μM [μM] 1

13 2

60 3

10 4

43 5

62 6

44 7

19 8

46 9

52 10

25 11

0.35 12

55 13

68 14

35 15

40 16

11 17

21 18

11 19

43 20

26 21

36 22

35 23

39 24

29 25

12 26

23 27

9 28

63 29

30 30

21 31

27 32

26 33

21 34

25 35

24 36

26 37

23 38

33 39

24 40

25 41

25 42

27 43

17 44

29 45

24All assays were repeated at least twice and representative results areshown.

In addition, bovine kidney heparan sulfate (Sigma Cat. No. H7640)conjugated to Bovine Serum Albumin (Sigma Cat. No. A7638) was used inthe in vitro assay as described herein above, to demonstrate L-selectinbinding to HS-GAGs different from heparin, and to demonstrate inhibitionof the binding by inhibitor compounds. FIG. 8 shows the inhibition ofL-selectin binding to bovine kidney heparan sulfate by several differentinhibitor compounds of formula I.

Example 6 Assays to Demonstrate Direct Interaction of InhibitorCompounds with Heparin and Other HS-GAGs

Assay 1.

In order to demonstrate that the L-selectin inhibitor Compounds indeedbind directly to heparin and other HS-GAGs, individual compounds wereincubated with immobilized heparin in the absence of L-selectin asfollows: 96 well ELISA plates were coated with Heparin-BSA, then blockedwith BSA as described in Example 5. Inhibitor Compounds at finalconcentration 0.1-200 μM were incubated in the ELISA plate for 90 min,and then washed with incubation buffer. After washing, L-selectin/IgGwas added to the wells pre-incubated with compounds. At the same time,in separate control wells, L-selectin was co-incubated with inhibitorCompounds for 90 min. Following the incubation, L-selectin bound to theplate was quantified by antibody conjugated to Horse Radish Peroxidaseand OD measurement as performed as described in Example 5. InhibitorCompounds No. 5 and No. 11 exerted the same level of inhibition onL-selectin binding to heparin in the pre-incubation or the co-incubationexperiments.

Assay 2.

Additional evidence for the direct binding of Inhibitor Compounds toheparin and other HS-GAGs was demonstrated by an extension of Assay 1.Ninety-six well ELISA plates were coated with Heparin-BSA, then blockedwith BSA as described above. Inhibitor Compounds, at concentrationspredetermined to inhibit L-selectin binding, were incubated in the ELISAplate for 60 min, and then washed with incubation buffer. After washing,L-selectin/IgG was added to the plate at increasing concentrations(5-250 μg/ml) and incubated for 90 minutes. Following the incubation,L-selectin bound to the plate was quantified by antibody conjugated toHorse Radish Peroxidase followed by optical density measurement asdescribed in Example 5. Quantitative L-selectin binding was demonstratedat the higher L-selectin concentrations (50-250 μg/ml), demonstratingthat the inhibitor Compounds prevented L-selectin binding to heparin,and therefore these results confirm that the inhibitor Compoundsdirectly interact with heparin, the latter is thus an L-selectinreceptor. An example is shown in FIG. 7. The dose response curvedelineated with open triangles (Control) represents the results obtainedafter incubation of the plate with L-selectin in the absence of theinhibitor Compound no. 1.

Example 7 Inhibition of Leukocyte Adhesion to Endothelial Cells UnderShear Flow by Inhibitor Compounds

Human T-lymphocytes were passed over a layer of human endothelial cellsaccording to the method of Lawrence and Springer (Cell 65: 859-873,1991). At high shear flow, migrating T-lymphocytes adhered transientlyand intermittently to the endothelial cells as a consequence ofHS-GAG-ECAM interaction. The resulting T-cell rolling was recorded byvideo camera and the number of rolling cells per defined area during aconstant period of time was determined. Soluble heparin (competitor ofcell surface GAGs) abolished T-cell rolling under high shear flowconditions. Compounds no. 1, 11 and 25, each at 25 μM, inhibited cellrolling by 90, 90 and 70%, respectively. Test Compound (TC) % rollingAssay Buffer >60 Heparin (5 μg/ml) 0 Compound no. 11 <10 Compound no. 25<10 Compound no. 1 30

Example 8 A Model of Leukocyte and Neutrophil Infiltration into MousePeritoneum

BALB/c mice (6 weeks old, ˜20 g in weight, 15 mice/group) receivedintraperitoneal injection of an inhibitor compound in 0.2 mlDMSO/Tween/sterile saline 1 hour before administration of thioglycollate(Sigma). Control groups received vehicle and sham controls received nothioglycollate. Mice were injected intraperitoneally with 1 ml of 3%thioglycollate broth (Xie, X. et al., J. Biol. Chem., 275, 34818-34825,2000). Mice were sacrificed after 3 hours, and the peritoneal cavitieswere ravaged with 5 μl of ice-cold saline containing 2 mM EDTA toprevent clotting. After red blood cell lysis, leukocytes were counted ina hemocytometer. Neutrophils were counted after staining with Türck.Data was expressed as mean±SEM, and statistical analysis was performedby Student t test. A value of P<0.05 was taken to denote statisticalsignificance.

Thioglycollate administration induced approximately 3-fold increase inleukocyte accumulation in the peritoneal cavity. Leukocyte migrationinto the peritoneal cavity was inhibited efficiently by administrationof inhibitor compounds including Compound No. 5 and Compound no. 11.Similar results were obtained when the neutrophil counts weredetermined. Compound no. 5 was tested in more detail at three doses: 2mg/kg, 10 mg/kg, and 50 mg/kg (FIG. 4). The compound was found to be apotent inhibitor of leukocyte migration; the infiltration was reduced by75% at a dose of 50 mg/kg, by 50% at 10 mg/kg, and by 25% at 2 mg/kg. Itis well known that leukocyte migration and infiltration in vivo is ahallmark of inflammatory, autoimmune and other disorders. The ability ofthe inhibitor compounds of the invention to inhibit leukocyteinfiltration in vivo has therefore therapeutic applications for thesedisorders.

Example 9 Carrageenan-Induced Paw Edema

Acute edema was induced in the left hind paw of Balb/c mice by injecting0.02 ml of freshly prepared solution of 2% carrageenan after 60 min oftest compound administration (Torres, S. R. et al., European Journal ofPharmacology 408: 199-211, 2000). The right paw received 0.02 ml ofsaline, which served as a control. Carrageenan was injected under theplantar region of right hind paw and the paw thickness was measured at2, 4 and 24 hours after carrageenan challenge using a Mitutoyoengineer's micrometer expressed as the difference between right and leftpad as mean±SEM. Inhibitor compounds significantly reduced carrageenaninduced paw edema after i.p. administration. A dose response curve forCompound no. 5 is shown in FIG. 5. These results demonstrate thatcompounds inhibiting GAG binding to GAG-ECAMs display anti-inflammatoryactivity.

Example 10 Delayed-Type Hypersensitivity (DTH)

Mice (15 animals per group) were sensitized by topical application of a2% oxazolone (4-ethoxymethylene-2-phenyl-2-oxazoline-5-one; Sigma, StLouis, Mo.) solution in acetone/olive oil (4:1 vol/vol) to shavedabdomen (50 μl) and to each paw (5 μl) (Lange-Asschenfeldt B. et al.,Blood 99:538-545, 2002). Five days after sensitization, right ears werechallenged by topical application of 10 μl of a 1% oxazolone solution,whereas left ears were treated with vehicle alone. Compounds wereadministered 1 hr prior to challenge. The extent of inflammation wasmeasured 24 hours after challenge, using the mouse ear-swelling test.The unpaired Student t test was used for statistical analyses.

As illustrated in FIG. 6, Compound no. 5 (at a dose of 3 mg/kg,administered iv) inhibited DTH to 56% of control value 24 hours afterchallenge. Data were statistically significant at p>0.001.

Example 11 Trinitrobenzine Sulfonic Acid (TNBS) Induced Colitis

Control mice (12 per group) were injected intraperitoneally (IP) withTest Compound (TC) Vehicle (Tween 80, 5%, 200 μl). Experimental mice (12per group) were injected IP with TC (10 mg/kg, or 35 mg/kg in 200 μl).The control and experimental mice were injected once per day for 7successive days. 24 hours after the first IP injection, InflammatoryBowel Disease (IBD) was induced in the control, experimental, and in anuntreated group by intra-rectal administration of TNBS (150 mg/kgdissolved in NaCl (0.9%): EtOH (50%) (1:1; 80 μl mouse). All of the micewere killed by cervical dislocation 7 days after TNBS administration.The colons of the mice were examined under a dissecting microscope (X5)to evaluate the macroscopic lesions on a scale of 0 to 10 (Colonicdamage score). As shown in FIG. 9, inhibitor Compound no. 11significantly inhibited the colonic damage at doses of 10 and 35 mg/kg.FIG. 10 shows the effect of oral administration of Compound no. 11 inthe same experimental model. The compound was administered once a dayfor 7 days in a suspension with 0.5% methylcellulose per os. There were12 mice per group and the control mice received 0.5% methylcelluloseonly. Compound no. 11 significantly inhibited the colonic damage atdoses of 3, 50 and 100 mg/kg per os (FIG. 10).

Example 12 Experimental Autoimmune Encephalomyelitis (EAE)

In autoimmune conditions, T cells reactive to self-antigens escapeelimination in the thymus and are activated in the periphery, where theycan provoke damage in specific organs. EAE, a model of autoimmunedisease that is induced in Lewis rats, bears many similarities to thehuman disease Multiple Sclerosis (MS). T cells found in brain lesions ofMS patients have TCR junctional rearrangements that are identical to Tcells found in the spinal cords of Lewis rats immunized with a peptideof myelin basic protein MBP p87-99. In addition, a major T and B cellresponse in MS patients is directed to MBP p87-99.

EAE is induced in rats by immunization with MBP p87-99. The inhibitorCompounds are administered once daily by intra-peritoneal injection for3 consecutive days starting 1 day before the appearance of EAE symptoms(day 12 after EAE induction). The degree of clinical disease is scoredas follows: 0=no signs; 1=loss of tail tonicity; 2=paralysis of hindlimbs; 3=paralysis of all four limbs 4=quadriplegic animal in a moribundstate. As shown in FIG. 11, Compound no. 11 significantly inhibitedclinical signs of EAE at a dose of 10 and 35 mg/kg.

Example 13 Inhibition of Growth of Cancer Cell Lines

Each cell line (MCF7 (Breast carcinoma), NCI-H460 (Non-small cell lungcarcinoma), and SF-268 (glioma) was pre-incubated on a microtiter plate.Inhibitor compounds were then added at a concentration of 0.1 μM and theculture incubated for 48 hours. End-point determinations were made withalamar blue (Gray G D, Wickstrom E, Biotechniques 21(5) 780-782, 1996).Results for each compound are presented as the percent of growth of thetreated cells as compared to the untreated cells. Compounds, whichreduce the growth of any one of the cell lines to approximately 32% orless were considered suitable for further and more extensive study(Monks A et al, J. Natl. Cancer Inst., 83:757-766, 1991. Compound no. 1inhibited the growth of the cell lines MCF7, NCI-H460, and SF-268 by99%, 100% and 99%, respectively.

Example 14 Inhibition of Leukemia Cell Growth

Each cell line (RPMI-8226 (Multiple Myeloma), MOLT-4 (Acutelymphoblastic leukemia), CCR-CEM (Human T Cell leukemia), K562 (Chronicmyeloid leukemia), SR (Chronic myeloid leukemia) was pre-incubated on amicrotiter plate (Monks A et al, J. Natl. Cancer Inst., 83:757-766,1991). Inhibitor compounds were then added at different concentrations(10⁻⁸-10⁻⁴ Molar) and the culture incubated for 48 hours. Asulforhodamine B (SRB) protein assay was used to estimate cell viabilityor growth (Skehan P et al, J. Natl. Cancer Inst., 82:1107-1112, 1990).Compound no. 1 inhibited the growth of the cell lines RPMI-8226, MOLT4,CCR-CEM, K562 and SR to 100%, 100%, 100%, 100% and 93%, respectively.There was negligible cell death (less than 15%).

Example 15 Methods to Measure the Counteracting Actions of Beparin andOther Anticoagulant GAGs on Coagulation

Assay 1. Methods for in vitro Determinations of the Effects of InhibitorCompound on Reversal of Factor Xa Activity.

Solutions of Lovenox (Rhone Poulenc Rohrer), Orgaran (Organan), orunfractionated heparin (Sigma) are prepared in 0.32% sodium citrate orin normal human plasma to contain 0.5 U/ml anti Factor Xa activity.Calibrations are made against the standards provided by the StachromHeparin (Diagnostica Stago) assay kit (Dignac M et al, Nouv. Res. FrHematol. 35:545-549, 1994). The heparin/ATIII complex is allowed to format 37° C. for 2 minutes, inhibitor Compound is added, the mixture isincubated for an additional 1-5 minutes, then Factor Xa is added, andfinally the chromogenic substrate is added for 1 minute, and theabsorbance is read at 405 nm. The increase in absorbance of theheparinized control vs. that of the test sample is divided by thedifference in the absorbance at 405 between the heparinized control andthe control without heparin to obtain the % reversal.

Assay 2. In vitro Effect of the Inhibitor Compound on Reversal ofInhibition of Thrombin Activity by Unfractionated Heparin.

Plasma is obtained from normal donors. Thrombin concentration (humanalpha thrombin, Enzyme Research Laboratories, South Bend, Ind.) isstandardized to produce a clotting time of 20-22 seconds. Heparin isadded at 0.5 IU anti-thrombin activity/ml. The clotting time for heparinalone is approximately 3 minutes. To test the effects of the compoundsin this system, one minute after addition of heparin to the plasma, thecompounds are added in concentrations ranging from 0.1-100 μM. After oneminute, thrombin is added and the clotting time is determined.

Assay 3. In vivo Effects of Inhibitor Compounds in Reversing Effects ofLovenox on Factor Xa Activity.

Rats (300-400 gm) are anesthetized with ketamine/acepromazine and arecannulated in the left jugular vein and right femoral vein. Blood isdrawn immediately before injection of Lovenox to establish baselineFactor Xa activity. Lovenox (43 IU anti-FXa activity/kg in 0.1 mlsaline, based on suggested dosage for humans) is injected through thejugular catheter, followed immediately by 0.2 ml of saline. Blood (0.1ml) is collected into sodium citrate from the femoral vein every 30seconds for 3 min. The compound is injected at 3 min through the jugularcatheter in 0.1 ml of phosphate-buffered saline, followed by a 0.2 mlsaline flush. Compounds are administered and blood collection isimmediately resumed every 30 seconds until 10 minutes after the initialLovenox injection, then at 15, 20, 25 and 30 min. The samples arecentrifuged to obtain plasma and are assayed for residual Lovenox byassay of anti-Factor Xa activity by the Stachrom Heparin test kit.Absorbance at 405 nm is measured after a 1-minute incubation with thechromogenic Factor Xa substrate.

It will be appreciated by persons skilled in the art that the presentinvention is not limited by what has been particularly shown anddescribed herein above. Rather the scope of the invention is defined bythe claims that follow.

1. A thieno[2,3-c]pyridine compound of the formula2-[[4-[[ethyl(phenylmethyl)amino]sulfonyl]benzoyl]amino]-6-ethyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxamide.2. A thieno[2,3-c]pyridine compound of the formula2-[[4-[(4-methyl-1-piperazinyl)sulfonyl]benzoyl]amino]-6-ethyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxamide.3. A pharmaceutical composition comprising as an active ingredient acompound of the general formula I:

wherein: R₁ is selected from the group consisting of H; straight orbranched alkyl of 1-6 carbon atoms; arylalkyl; substituted arylalkyl;cycloalkyl, optionally substituted with alkyl groups; alkanoyl;arylcarbonyl optionally substituted at the aryl group;cycloalkylcarbonyl; alkoxycarbonyl; R₂ is selected from the groupconsisting of carboxy; cyano; aminocarbonyl; alkylaminocarbonyl;arylaminocarbonyl optionally substituted at the aryl group;dialkylaminocarbonyl wherein each alkyl is straight or branched chainC₁-C₆ alkyl or both alkyl groups together may form a 3-7 memberedsaturated, unsaturated or aromatic monocyclic or bicyclic nitrogencontaining heterocyclyl, optionally containing one or two additionalheteroatoms; alkoxycarbonyl; alkanoyl; cycloalkylcarbonyl; arylcarbonyloptionally substituted on the aryl group, benzothiazol-2-yl; R₃ and R₄are selected from the group consisting of C₁-C₆ alkyl, optionallysubstituted by hydroxy, alkoxy, amino or alkylamino, C₂-C₄monounsaturated alkenyl, cycloalkyl, aryl, arylmethyl, or R₃ and R₄together may form an optionally substituted 5-7 membered saturated,unsaturated or aromatic monocyclic or bicyclic nitrogen containingheterocyclyl, optionally containing one or two additional heteroatoms;R₅, R₆, R₇ and R₈ are selected from the group consisting of H or C₁-C₆alkyl, with the proviso that when R₅, R₆, R₇ and R₈ are C₁-C₆ alkyl, R₁is hydrogen; and pharmaceutically acceptable salts thereof; furthercomprising a pharmaceutically acceptable diluent or carrier.
 4. Thepharmaceutical composition according to claim 3, wherein R₁ is selectedfrom the group consisting of methyl, ethyl, 1-methylethyl, phenylmethyl,acetyl, ethoxycarbonyl and R₅═R₆═R₇═R₈ are hydrogens.
 5. Thepharmaceutical composition according to claim 3, wherein R₁ is hydrogenand R₅═R₆═R₇═R₈ are hydrogens or methyl groups.
 6. The pharmaceuticalcomposition according to claim 3, wherein R₁═R₅═R₆ is methyl and R₇═R₈are hydrogens.
 7. The pharmaceutical composition according to claim 3,wherein R₂ is selected from the group consisting of cyano,methoxycarbonyl, ethoxycarbonyl, aminocarbonyl, methylaminocarbonyl,dimethylaminocarbonyl, pyrrolidinylcarbonyl, piperidinylcarbonyl,morpholinylcarbonyl, benzothiazol-2-yl.
 8. The pharmaceuticalcomposition according to claim 3, wherein R₃ and R₄ are selected fromthe group consisting of methyl, ethyl, propyl, butyl, methoxyethyl,chlorobutyl, cyanoethyl, phenyl, cyclopentyl, cyclohexyl, phenylmethyl,allyl or crotyl, R₃ and R₄ may be equal or different.
 9. Thepharmaceutical composition according to claim 3, wherein R₃ and R₄ formpyrrolidine, piperidine, 2-methyl, 3-methyl, 4-methyl or 3,5-dimethylpiperidine, perhydroazepine, morpholine, piperazine, 4-methylpiperazine,3,4-dihydro-2(1H)-isoquinolinyl, 3,4-dihydro-1(2H)quinoline,1,3,3-trimethyl-6-azabicyclo[3.2.1]oct-6-ane and substituted derivativesthereof.
 10. The pharmaceutical composition according to claim 3 whereinthe compound of Formula I is selected from:2-[[4-[(ethylbutylamino)sulfonyl]benzoyl]amino]-3-(benzothiazol-2-yl)-6-ethyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine;2-[[(4-(3,4-dihydro-2(1H)-isoquinolinyl)sulfonyl]benzoyl]amino]-6-(1-methylethyl)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxamide;2-[[4-(methylphenylamino)sulfonyl]benzoyl]amino]-6-(1-methylethyl)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxamide;2-[[4-(3,4-dihydro-2(1H)-isoquinolinyl)sulfonyl]benzoyl]amino]-4,5,6,7-tetrahydro-5,5,7,7-tetramethylthieno[2,3-c]pyridine-3-carboxamide;2-[[4-[(diethylamino)sulfonyl]benzoyl]amino]-3-(benzothiazol-2-yl)-6-ethyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine;2-[[4-(morpholinylsulfonyl)benzoyl]amino]-3-(benzothiazol-2-yl)-6-(l-methylethyl)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine;2-[[4-(diethylamino)sulfonyl]benzoyl]amino]-4,5,6,7-tetrahydro-5,5,7,7-tetramethylthieno[2,3-c]pyridine-3-carboxylic acid ethyl ester;2-[[4-(3,4-dihydro-1(2H)-quinolinyl)sulfonyl]benzoyl)amino]-3-(benzothiazol-2-yl)-4,5,6,7-tetrahydro-5,5,7,7-tetramethylthieno[2,3-c]pyridine;2-[[4-(hexahydro-1H-azepin-1-yl)sulfonyl]benzoyl]amino]-4,5,6,7-tetrahydro-5,5,7,7-tetramethylthieno[2,3-c]pyridine-3-carboxylic acid ethyl ester;2-[[4-[[4-(methyl)-1-piperazinyl]sulfonyl]benzoyl]amino]-3-(benzothiazol-2-yl)-6-methyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine;2-[[4-[(1,3,3-trimethyl-6-azabicyclo[3.2.1]oct-6-yl)sulfonyl]benzoyl]amino]-3-(benzothiazol-2-yl)-6-methyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine;2-[[4-[(methylphenylamino)sulfonyl]benzoyl]amino]-3-(benzothiazol-2-yl)-6-methyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine;2-[[4-(morpholinylsulfonyl)benzoyl]amino]-3-(benzothiazol-2-yl)-6-methyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine;2-[[4-[(diethylamino)sulfonyl]benzoyl]amino]-3-(benzothiazol-2-yl)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-6-carboxylicacid ethyl ester;2-[[4-[[4-(3-methyl-1-piperidinyl)]sulfonyl]benzoyl]amino]-3-(benzothiazol-2-yl)-6-methyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine;2-[[4-[(diethylamino)sulfonyl]benzoyl]amino]-3-(benzothiazol-2-yl)-6-(phenylmethyl)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine;2-[[4-[(diethylamino)sulfonyl]benzoyl]amino]-3-(benzothiazol-2-yl)-6-methyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine;2-[[4-[[4-(ethoxycarbonyl)-1-piperazinyl]sulfonyl]benzoyl]amino]-4,5,6,7-tetrahydro-5,5,7,7-tetramethylthieno[2,3-c]pyridine-3-carboxamide;2-[[4-[(cyclohxylmethylamino)sulfonyl]benzoyl]amino]-4,5,6,7-tetrahydro-5,5,7,7-tetramethylthieno[2,3-c]pyridine-3-carboxamide;2-[[4-[(di-2-propenylamino)sulfonyl]benzoyl]-4,5,6,7-tetrahydro-5,5,7,7-tetramethylthieno[2,3-c]pyridine-3-carboxylicacid methyl ester;2-[[4-[(di-2-methoxyethylamino)]sulfonyl]benzoyl]-4,5,6,7-tetrahydro-5,5,7,7-tetramethylthieno[2,3-c]pyridine-3-carboxamide;2-[[4-[(1,3,3-trimethyl-6-azabicyclo[3.2.1.]oct-6-yl)sulfonyl]benzoyl]amino]-6-methyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxamide;2-[[4-[(diethylamino)sulfonyl]benzoyl]amino]-3-(benzothiazol-2-yl)-6-methyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine;2-[[4-[(diethylamino)sulfonyl]benzoyl]amino]-3-(benzothiazol-2-yl)-6-(1-methylethyl)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine;2-[[4-[(di-2-methoxyethylamino)sulfonyl]benzoyl]-amino]-3-(benzothiazol-2-yl)-6-methyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine;2-[[4-[(methylphenylamino)sulfonyl]benzoyl]amino]-3-(benzothiazol-2-yl)-6-methyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine;2-[[4-[[4-(ethoxycarbonyl)-1-piperazinyl]sulfonyl]benzoyl]amino]-3-(benzothiazol-2-yl)-6-methyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine;2-[[4-[(methylbutylamino)sulfonyl]benzoyl]amino]-6-(1-methylethyl)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxylicacid ethyl ester;2-[[4-[[4-(ethoxycarbonyl)-1-piperazinylasulfonyl]benzoyl]amino]-6-(1-methylethyl)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxylicacid ethyl ester;2-[[4-(diethylamino)sulfonyl]benzoyl]amino]-4,5,6,7-tetrahydro-5,5,7,7-tetramethylthieno[2,3-c]pyridine-3-carboxamide;2-[[4-[(methylphenylamino)sulfonyl]benzoyl]amino]-6-ethyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxylicacid methylamide;2-[[4-[[ethyl(phenylmethyl)amino]sulfonyl]benzoyl]amino]-6-ethyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxamide;2-[[4-[(4-methyl-1-piperazinyl)sulfonyl]benzoyl]amino]-6-ethyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxamide;2-[[(4-(3,4-dihydro-1(2H)-quinolinyl)sulfonyl]benzoyl]amino]-6-ethyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxylicacid methylamide;2-[[4-[(4-methyl-1-piperazinyl)sulfonyl]benzoyl]amino]-6-ethyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxylicacid methylamide;2-[[4-[(4-methyl-l-piperazinyl)sulfonyl]benzoyl]amino]-3-(benzothiazol-2-yl)-6-ethyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine;2-[[4-(diethylamino)sulfonyl]benzoyl]amino]-6-ethyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxylicacid N-methylamide;2-[[4-(diethylamino)sulfonyl]benzoyl]amino]-6-ethyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxylicacid morpholinylamide.
 11. The pharmaceutical composition according toclaim 10 wherein the compound of formula I is:2-[[4-[(ethylbutylamino)sulfonyl]benzoyl]amino]-3-(benzothiazol-2-yl)-6-ethyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine;12. The pharmaceutical composition according to claim 10 wherein thecompound of formula I is:2-[[4-[(diethylamino)sulfonyl]benzoyl]amino]-3-(benzothiazol-2-yl)-6-ethyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine;13. The pharmaceutical composition according to claim 10 wherein thecompound of formula I is:2-[[4-[[ethyl(phenylmethyl)amino]sulfonyl]benzoyl]amino]-6-ethyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxamide.14. The pharmaceutical composition according to claim 10 wherein thecompound of formula I is:2-[[4-[(4-methyl-1-piperazinyl)sulfonyl]benzoyl]amino]-6-ethyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxamide.15. The pharmaceutical composition according to claim 3 capable ofinhibiting the interaction of GAGs with GAG specific ECAMs.
 16. Thepharmaceutical composition according to claim 15 wherein the GAG isselected from the group consisting of heparan sulfate (HS-GAG), heparin,chondroitin sulfate, dermatan sulfate, keratan sulfate and derivativesand fragments thereof.
 17. The pharmaceutical composition according toclaim 16 wherein the GAG is HS-GAG.
 18. The pharmaceutical compositionaccording to claim 15 wherein the GAG specific ECAMs are selected fromthe group consisting of L-selectin and P-selectin.
 19. Thepharmaceutical composition according to claim 15 for inhibition ofneutrophil infiltration in vivo, with the proviso that the compound isother than2-[[4-[(1,3,3-trimethyl-6-azabicyclo[3.2.1.]oct-6-yl)sulfonyl]benzoyl]amino]-6-ethyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxylicacid ethyl ester.
 20. A method for inhibiting cell adhesion or cellmigration in vitro comprising the step of exposing the cells to apharmaceutical composition according to claim 3 in an amount sufficientfor preventing the interactions of the GAG with at least one GAGspecific ECAM.
 21. A method for the treatment or prevention of diseasesor disorders related to cell adhesion or cell migration mediated byGAG-ECAM interactions, comprising the step of administering to a subjectin need thereof a therapeutically effective amount of a pharmaceuticalcomposition comprising a compound of the general formula I:

wherein: R₁ is selected from the group consisting of H; straight orbranched alkyl of 1-6 carbon atoms; arylalkyl; substituted arylalkyl;cycloalkyl, optionally substituted with lower alkyl groups; loweralkanoyl; arylcarbonyl optionally substituted at the aryl group;cycloalkylcarbonyl; alkoxycarbonyl; R₂ is selected from the groupconsisting of carboxy; cyano; aminocarbonyl; alkylaminocarbonyl;arylaminocarbonyl optionally substituted at the aryl group;dialkylaminocarbonyl wherein each alkyl is straight or branched chainlower alkyl or both alkyl groups together may form a 3-7 memberedsaturated, unsaturated or aromatic monocyclic or bicyclic nitrogencontaining heterocyclyl, optionally containing one or two additionalheteroatoms; alkoxycarbonyl; lower alkanoyl; cycloalkylcarbonyl;arylcarbonyl optionally substituted on the aryl group,benzothiazol-2-yl; R₃ and R₄ are selected from the group consisting ofC₁-C₆ alkyl, optionally substituted by hydroxy, alkoxy, amino oralkylamino, C₂-C₄ monounsaturated alkenyl, cycloalkyl, aryl, arylmethyl,or R₃ and R₄ together may form an optionally substituted 5-7 memberedsaturated, unsaturated or aromatic monocyclic or bicyclic nitrogencontaining heterocyclyl, optionally containing one or two additionalheteroatoms; R₅, R₆, R₇ and R₈ are selected from the group consisting ofH or C₁-C₆ alkyl, with the proviso that when R₅, R₆, R₇ and R₈ are C₁-C₆alkyl, R₁ is hydrogen; and pharmaceutically acceptable salts thereof;further comprising a pharmaceutically acceptable diluent or carrier. 22.The method according to claim 21 wherein R₁ is selected from the groupconsisting of methyl, ethyl, 1-methylethyl, phenylmethyl, acetyl,ethoxycarbonyl and R₅═R₆═R₇═R₈ are hydrogens.
 23. The method accordingto claim 21 wherein R₁ is hydrogen and R₅═R₆═R₇═R₈ are hydrogens ormethyl groups.
 24. The method according to claim 21 wherein R₁═R₅═R₆ ismethyl and R₇═R₈ are hydrogens.
 25. The method according to claim 21wherein R₂ is selected from the group consisting of cyano,methoxycarbonyl, ethoxycarbonyl, aminocarbonyl, methylaminocarbonyl,dimethylaminocarbonyl, pyrrolidinylcarbonyl, piperidinylcarbonyl,morpholinylcarbonyl, (3,5-dimethyl-1H-pyrazolyl) carbonyl,benzothiazol-2-yl.
 26. The method according to claim 21 wherein R₃ andR₄ are selected from the group consisting of methyl, ethyl, propyl,butyl, methoxyethyl, chlorobutyl, cyanoethyl, phenyl, cyclopentyl,cyclohexyl, phenylmethyl, allyl or crotyl, R₃ and R₄ may be equal ordifferent.
 27. The method according to claim 21 wherein R₃ and R₄ formpyrrolidine, piperidine, 2-methyl, 3-methyl, 4-methyl or 3,5-dimethylpiperidine, perhydroazepine, morpholine, piperazine, 4-methylpiperazine,3,4-dihydro-2(1H)-isoquinolinyl, 3,4-dihydro-1(2H)quinoline,1,3,3-trimethyl-6-azabicyclo[3.2.1]oct-6-ane and substituted derivativesthereof.
 28. The method according to claim 21 wherein the compound offormula I is selected from:2-[[4-[(ethylbutylamino)sulfonyl]benzoyl]amino]-3-(benzothiazol-2-yl)-6-ethyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine;2-[[(4-(3,4-dihydro-2(1H)-isoquinolinyl)sulfonyl]benzoyl]amino]-6-(l-methylethyl)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxamide;2-[[4-(methylphenylamino)sulfonyl]benzoyl]amino]-6-(1-methylethyl)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxamide;2-[[4-(3,4-dihydro-1(2H)-quinolinyl)sulfonyl]benzoyl]amino]-4,5,6,7-tetrahydro-5,5,7,7-tetramethylthieno[2,3-c]pyridine-3-carboxamide;2-[[4-[(diethylamino)sulfonyl]benzoyl]amino]-3-(benzothiazol-2-yl)-6-ethyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine;2-[[4-(morpholinylsulfonyl)benzoyl]amino]-3-(benzothiazol-2-yl)-6-(1-methylethyl)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine;2-[[4-(diethylamino)sulfonyl]benzoyl]amino]-4,5,6,7-tetrahydro-5,5,7,7-tetramethylthieno[2,3-c]pyridine-3-carboxylic acid ethyl ester;2-[[4-(3,4-dihydro-1(2H)-quinolinyl)sulfonyl]benzoyl]amino]-3-(benzothiazol-2-yl)-4,5,6,7-tetrahydro-5,5,7,7-tetramethylthieno[2,3-c]pyridine;2-[[4-(hexahydro-1H-azepin-1-yl)sulfonyl]benzoyl]amino]-4,5,6,7-tetrahydro-5,5,7,7-tetramethylthieno[2,3-c]pyridine-3-carboxylic acid ethyl ester;2-[[4-[[4-(methyl)-1-piperazinyl]sulfonyl]benzoyl]amino]-3-(benzothiazol-2-yl)-6-methyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine;2-[[4-[(1,3,3-trimethyl-6-azabicyclo[3.2.l]oct-6-yl)sulfonyl]benzoyl]amino]-3-(benzothiazol-2-yl)-6-methyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine;2-[[4-[(methylphenylamino)sulfonyl]benzoyl]amino]-3-(benzothiazol-2-yl)-6-methyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine;2-[[4-(morpholinylsulfonyl)benzoyl]amino]-3-(benzothiazol-2-yl)-6-methyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine;2-[[4-[(diethylamino)sulfonyl]benzoyl]amino]-3-(benzothiazol-2-yl)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-6-carboxylicacid ethyl ester;2-[[4-[[4-(3-methyl-1-piperidinyl)]sulfonyl]benzoyl]amino]-3-(benzothiazol-2-yl)-6-methyl4,5,6,7-tetrahydro thieno[2,3-c]pyridine;2-[[4-[(diethylamino)sulfonyl]benzoyl]amino]-3-(benzothiazol-2-yl)-6-(phenylmethyl)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine;2-[[4-[(diethylamino)sulfonyl]benzoyl]amino]-3-(benzothiazol-2-yl)-6-methyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine;2-[[4-[[4-(ethoxycarbonyl)-1-piperazinyl]sulfonyl]benzoyl]amino]-4,5,6,7-tetrahydro-5,5,7,7-tetramethylthieno[2,3-c]pyridine-3-carboxamide;2-[[4-[(cyclohxylmethylamino)sulfonyl]benzoyl]amino]-4,5,6,7-tetrahydro-5,5,7,7-tetramethylthieno[2,3-c]pyridine-3-carboxamide;2-[[4-[(di-2-propenylamino)sulfonyl]benzoyl]-4,5,6,7-tetrahydro-5,5,7,7-tetramethylthieno[2,3-c]pyridine-3-carboxylicacid methyl ester;2-[[4-[(di-2-methoxyethylamino)]sulfonyl]benzoyl]-4,5,6,7-tetrahydro-5,5,7,7-tetramethylthieno[2,3-c]pyridine-3-carboxamide;2-[[4-[(l,3,3-trimethyl-6-azabicyclo[3.2.l]oct-6-yl)sulfonyl]benzoyl]amino]-6-methyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxamide;2-[[4-[(diethylamino)sulfonyl]benzoyl]amino]-3-(benzothiazol-2-yl)-6-methyl-4,5,6,7-tetrahydrothieno[2,3-c)pyridine;2-[[4-[(diethylamino)sulfonyl]benzoyl]amino]-3-(benzothiazol-2-yl)-6-(1-methylethyl)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine;2-[[4-[(di-2-methoxyethylamino)sulfonyl]benzoyl]-amino]-3-(benzothiazol-2-yl)-6-methyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine;2-[[4-[(methylphenylamino)sulfonyl]benzoyl]amino]-3-(benzothiazol-2-yl)-6-methyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine;2-[[4-[[4-(ethoxycarbonyl)-1-piperazinyl]sulfonyl]benzoyl]amino]-3-(benzothiazol-2-yl)-6-methyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine;2-[[4-[(methylbutylamino)sulfonyl]benzoyl]amino]-6-(1-methylethyl)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxylicacid ethyl ester;2-[[4-[[4-(ethoxycarbonyl)-1-piperazinyl]sulfonyl]benzoyl]amino]-6-(1-methylethyl)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxylicacid ethyl ester;2-[[4-(diethylamino)sulfonyl]benzoyl]amino]-4,5,6,7-tetrahydro-5,5,7,7-tetramethylthieno[2,3-c]pyridine-3-carboxamide;2-[[4-[(methylphenylamino)sulfonyl]benzoyl]amino]-6-ethyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxylicacid methylamide;2-[[4-[[ethyl(phenylmethyl)amino]sulfonyl]benzoyl]amino]-6-ethyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxamide;2-[[4-[(4-methyl-1-piperazinyl)sulfonyl]benzoyl]amino]-6-ethyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxamide;2-[[(4-(3,4-dihydro-1(2H)-quinolinyl)sulfonyl]benzoyl]amino]-6-ethyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxylicacid methylamide;2-[[4-[(4-methyl-1-piperazinyl)sulfonyl]benzoyl]amino]-6-ethyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxylicacid methylamide;2-[[4-[(4-methyl-1-piperazinyl)sulfonyl]benzoyl]amino]-3-(benzothiazol-2-yl)-6-ethyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine;2-[[4-(diethylamino)sulfonyl]benzoyl]amino]-6-ethyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxylicacid N-methylamide;2-[[4-(diethylamino)sulfonyl]benzoyl]amino]-6-ethyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxylicacid morpholinylamide.
 29. The method according to claim 28 wherein thecompound of formula I is:2-[[4-[(ethylbutylamino)sulfonyl]benzoyl]amino]-3-(benzothiazol-2-yl)-6-ethyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine;30. The method according to claim 28 wherein the compound of formula Iis:2-[[4-[(diethylamino)sulfonyl]benzoyl]amino]-3-(benzothiazol-2-yl)-6-ethyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine;31. The method according to claim 28 wherein the compound of formula Iis:2-[[4-[[ethyl(phenylmethyl)amino]sulfonyl]benzoyl]amino]-6-ethyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxamide.32. The method according to claim 28 wherein the compound of formula Iis:2-[[4-[(4-methyl-1-piperazinyl)sulfonyl]benzoyl]amino]-6-ethyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxamide.33. The method according to claim 21 wherein the disease or disorderrelated to cell adhesion or cell migration is selected from the groupconsisting of an inflammatory process, an autoimmune process or disease,platelet-mediated pathologies, tumor metastasis, viral diseases,atherosclerosis, amyloid disorders, and kidney disease.
 34. The methodaccording to claim 33 wherein the inflammatory disorder is selected fromthe group consisting of septic shock, post-ischemic leukocyte-mediatedtissue damage, frost-bite injury or shock, acute leukocyte-mediated lunginjury, acute pancreatitis, asthma, traumatic shock, stroke, traumaticbrain injury, nephritis, acute and chronic inflammation, atopicdermatitis, psoriasis, uveitis, and retinitis, and inflammatory boweldisease.
 35. The method according to claim 33 wherein the autoimmunedisease is selected from the group consisting of rheumatoid arthritisand multiple sclerosis.
 36. A method of treatment or prevention of GAGmediated diseases or disorders comprising the step of administering to asubject in need thereof a therapeutically effective amount of apharmaceutical composition according to claim
 3. 37. The methodaccording to claim 36 wherein the GAG is HS-GAG.
 38. The methodaccording to claim 36 wherein the disease or disorder is selected fromthe group consisting of amyloid disorders, viral diseases, bacterialinfections, kidney diseases, cancer, tumor metastasis, and coagulationdisorders.
 39. The method according to claim 38 wherein the disease ordisorder is selected from the group consisting of Alzheimer's disease,type II diabetes, hepatitis C, hepatitis B, influenza, rhinovirusinfections, cytomegalovirus infections, AIDS, respiratory syncytialvirus infections, malaria, and leukemia.
 40. A method for modulatinganticoagulant activity of glycosaminoglycans in a subject comprising thestep of administering a therapeutically effective amount of apharmaceutical composition according to claim 3, thereby modulating theanticoagulant activity of glycosaminoglycans.
 41. The method accordingto claim 40, wherein the glycosaminoglycan is heparin.
 42. A method forthe prevention or treatment of inflammatory bowel disease comprising thestep of administering to a subject in need thereof a therapeuticallyeffective amount of a pharmaceutical composition comprising as an activeingredient a thieno[2,3-c]pyridine compound of formula2-[[4-[(diethylamino)sulfonyl]benzoyl]amino]-3-(benzothiazol-2-yl)-6-ethyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridineand a pharmaceutically acceptable salt thereof; further comprising apharmaceutically acceptable carrier or diluent.
 43. A method for theprevention or treatment of multiple sclerosis comprising the step ofadministering to a subject in need thereof a therapeutically effectiveamount of a pharmaceutical composition comprising as an activeingredient a thieno[2,3-c]pyridine compound of formula2-[[4-[(diethylamino)sulfonyl]benzoyl]amino]-3-(benzothiazol-2-yl)-6-ethyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridineand a pharmaceutically acceptable salt thereof; further comprising apharmaceutically acceptable carrier or diluent.